Term
| What did (Griffith, 1928) find out about DNA? |
|
Definition
| He found the transforming DNA that was used in the mouse experiment where the mouse died with the heat-treated deadly bacteria and the innate smooth bacteria but he did not know how it worked |
|
|
Term
| What did the Macleod, Avery and McCarty find about DNA? |
|
Definition
They found that with the mice in the Griffith experiment, it was DNA transformation that was to blame for this process.
They discovered the transformation mechanism |
|
|
Term
| What was the Hershey Chase experiment in 1952? |
|
Definition
| This was the experiment that proved that nucleic acid is the genetic material It used labelled protein or DNA and then used bacteriophages that will insert DNA into the bacteria. They then mixed the phages with the bacteria and found where the label ended up. It found the labelled DNA was in the bacteria therefore DNA was genetic material responsible for the phage reproduction |
|
|
Term
|
Definition
Phosphodiester bonds.
This is one of the oxygens on the phosphate end causing a precipitation reaction with the OH on the 3' carbon in the pentose ring |
|
|
Term
| Which direction does DNA bind? |
|
Definition
ALWAYS 5' TO 3'
DNA will bind onto the 3' end and then it will elongate this way |
|
|
Term
| What is the DNA structure in a cell? |
|
Definition
It is DNA wrapping around a histone protein made up of 6 subunits
They then form 'beads on a string' which will be packed |
|
|
Term
| What is the Meselson & Stahl experiment? |
|
Definition
| This is the experiment that was to prove semi conservative replication using the different molecular weights of Nitrogen and they they found that you get two bands in the replicated DNA from two different weights as they DNA was including the DNA from template and new strands |
|
|
Term
| What is an open reading frame? |
|
Definition
The ORF is the gene part of the DNA, it is the genetic information part of the DNA that will be transcribed into a protein.
The ORF is aided by all the transcription factors such as: enhancer, repressor, promoter an terminator |
|
|
Term
| What are the transcriptional products of the lac Operon? |
|
Definition
Beta-galactosidase
Lactose permease
Thiogalactosidase transacetylase |
|
|
Term
| What does allolactose do in the lac operon? |
|
Definition
| It acts as a small molecule inducer as it will bind to the allosteric site of the lac repressor protein and stop it from binding to the operator region and this will then cause ß-galactosidase and the other enzymes to be transcribed |
|
|
Term
| How does the lac operon work? |
|
Definition
No lactose
A repressor binds to the operating region and this will prevent the polymerase from binding to the operating region and therefore stop it from transcribing the gene product.
In presence of lactose
The lactose will bind to the repressor in its allosteric sites and they will then prevent the repressor from binding to the operating region. This allows the promoter region to stimulate the polymerase to move on to the genes and then transcribe the relevant RNA for the digestion of the lactose |
|
|
Term
|
Definition
DNA-binding domain
It is present in with the Activation domain complex that moves along the DNA and will act to recruit the large polymerase enzymes |
|
|
Term
| What is the RNA polymerase II holoenzyme? |
|
Definition
It is a complex made up of RNA Pol II and then other factors such as:
TATA box binding protein
Basal transcription factors (general transcription factors) - the parts that bind to the promoter region to initiate transcription |
|
|
Term
|
Definition
This is a part of the promoter region of genes in eukaryotes and archaea.
It is found in about a quarter of human genome promoter regions |
|
|
Term
|
Definition
This is the addition of multiple Adenosine bases on the 3' end.
It is stop degradation of the RNA molecule when transporting and such |
|
|
Term
|
Definition
The Untranslated Region
There can be the 5' prime UTR or 3' prime UTR. They are both used for controlling transcription factors and other factors during the transcription process |
|
|
Term
| What is a sense strand and an antisense strand? |
|
Definition
The sense strand is the DNA strand that has the same sequence as the RNA strand
The antisense strand is the strand that the RNA uses as its template |
|
|
Term
|
Definition
| This is for the prevention of degradation, regulation of nuclear export and promotion of the translation |
|
|
Term
| What is a major reason for the extreme ratio difference between mouse and human Mb:Genes? |
|
Definition
Mice have more genes because they have an extremely high number of genes which coded for their extremely sensitive sense of smell
Mice = 2300Mb - 21,000 genes
Human = 3200Mb - 20,000 genes |
|
|
Term
| How does alternate splicing aid the difference between males and females? |
|
Definition
Alternate splicing will induce a difference in genes that is controlled by the difference in the x and y chromosome.
This leads to repression of female or male specific genes |
|
|
Term
|
Definition
Both are restriction enzymes that will create a sticky end.
EcoRI = 5' sticky end
PstI = 3' sticky end |
|
|
Term
|
Definition
| These are both restriction enzymes that will create a blunt end |
|
|
Term
|
Definition
| It is an enzyme that will make a new phosphodiester bond and will stick complementary sticky ends or any blunt ends together |
|
|
Term
| How do transform a vector into a piece of non linear DNA? |
|
Definition
| You treat it with a restriction enzyme such as EcoRI, EcoRV or PstI and then you treat it with Phosphatase and this will mean that the phosphate groups on the open nucleotides are removed and then no phosphodiester bond can be formed and therefore the vector cannot return to circular |
|
|
Term
| How do you insert a piece of DNA into a vector? |
|
Definition
| Cut with restriction enzyme, treat with phosphatase, cut the inserting piece of DNA with the same restriction enzyme so there are complementary sticky ends and then mix the open vector with the DNA and then treat with ligase |
|
|
Term
| What was the first cloning vector? |
|
Definition
| It was pBR322. Made in 1977 by Bolivar et al., the vector contained origin of replication (ori and rop sites) for ColE1, two antibiotic resistance genes (AMPr and TETr) and unique restriction sites which were in the middle of either the AMPr or TETr genes |
|
|
Term
| How do you work out if the pBR322 vector has been successfully transformed and cloned? |
|
Definition
| You can cultivate it on a dish with tetracycline and ampicillin and if the transformation was successful, the gene will be inserted inside the TETr gene so it will not grow on TET plate but will on the AMP plate |
|
|
Term
| What are the three main methods for getting DNA into a bacterial cell? |
|
Definition
1) CaCl2 to create holes in bacterial membrane and then heat shock
2) Electroporation creates pores in the bacterial membrane
3) Gene gun will put a DNA coated bead straight into the bacterial cell mechanically |
|
|
Term
| What are the common marker genes for bacterial plasmids? |
|
Definition
Ampicillin
Tetracycline
Chloramphenicol
Kanamycin and neomycin
Bleomycin and zeocin
Hygromycin B |
|
|
Term
| How do you isolate plasmid DNA in a simple old fashioned description? |
|
Definition
| You can lyse the bacteria and the centrifuge it using a CsCl gradient to split the plasmid DNA with other bits |
|
|
Term
| What is the old fashioned way to isolate Plasmid DNA? |
|
Definition
You centrifuge the matter and the split occurs simply because go the density of the material in the centrifuge container. This is called isopycnic centrifugation and uses the CsCl gradient.
It was expensive and tedious to have to do over and over |
|
|
Term
| What is the modern way to isolate plasmid DNA? |
|
Definition
Treat with SDS to ensure an alkaline pH and then the SDS will disrupt cell membrane and the proteins within the cell. The plasmid DNA remains in solution however.
Then neutralise with potassium acetate and then you can centrifuge more easily and remove all the other precipitate to left with the plasmid DNA in the bottom.
Then drop through a silica or +vely charge ion exchange matrix column. plasmid DNA binds and then you can wash away any other impurities. Then just wash through with buffer and gather our plasmid DNA |
|
|
Term
| What did (Birnboim & Doly, 1979) discover to aid Plasmid DNA isolation? |
|
Definition
| They created a way to ensure that the DNA was at an alkaline pH so that it could be treated to break down the other matter and not the DNA by treating it with Sodium dodecyl sulphate (SDS) in the presence of NaOH |
|
|
Term
| What are the conditions for purification of plasmid DNA in a positively charge ion exchange column? |
|
Definition
Plasmid DNA will bind to the matrix in HIGH salt conditions so other precipitate can be washed away
Pure plasmid DNA can then be washed through with LOW salt conditions to obtain the pure DNA |
|
|
Term
| Which way does DNA move in the electrophoresis gel? |
|
Definition
| It moves towards the positively charged end as the DNA is negatively charge. |
|
|
Term
| What is Southern blotting? |
|
Definition
It is when you take a film and imprint onto it the results from a specific electrophoresis test. You then add a labelled DNA probe onto the film with a known sequence, complementary to the one you are looking for. You then allow to hybridise and then where the DNA probe goes, that is the DNA fragment you are looking for.
Northern blotting is the same but with RNA |
|
|
Term
| What is Western blotting? |
|
Definition
| This is using antibodies to find a specific protein. Can be done anywhere and does not use electrophoresis as it is not for DNA |
|
|
Term
| What is Northern blotting? |
|
Definition
| The same as Southern blotting but with RNA instead of DNA |
|
|
Term
| What is South-Western blotting? |
|
Definition
| This is using antibodies, like in Western blotting, for finding specific DNA fragment, like in Southern blotting. You use it to identify the proteins that will bind with specific DNA fragments by using labelled antibodies. |
|
|
Term
|
Definition
It describes an open reading frame.
This is region that will be transcribed and translated into a protein.
That and all the transcriptional control elements (enhancer, repressor, promoter, terminator etc.) |
|
|
Term
| How many genes in a human genome? |
|
Definition
|
|
Term
| How many genes in a mouse genome? |
|
Definition
|
|
Term
| What are the key concepts of vectors? |
|
Definition
They autonomously replicate and can carry foreign DNA
Can be isolated in quantities big enough for most laboratory manipulations
Based on naturally occurring DNA sequences but are modified to serve a number of particular functions
Different vectors are able to carry different sizes of foreign DNA
They can produce single strand DNA, have high level of protein encoding genes, produce RNA |
|
|
Term
|
Definition
Yeast artificial chromosome
Used before BACs to insert 100-1000kb of DNA into to be cloned and then mapped. |
|
|
Term
|
Definition
Bacterial artificial chromosome
Based on the functional Fertility (F) plasmid and is used for transforming and cloning bacteria
Insert size is around 150-350kb in size |
|
|
Term
|
Definition
P1 artificial chromosome
Used from the P1 bacteriophage
Can carry between 100-300kb in size
Used for cloning DNA in E. coli cells |
|
|
Term
|
Definition
Human artificial chromosome
Same as the BACs, PACs and YACs but for human cells or eukaryotes cells
About 5-6Mb in size which is much smaller but can be used to introduce new genes into humans |
|
|
Term
| What are the five main class of plasmid? |
|
Definition
F plasmid - Fertility Plasmid, capable of conjugation and the stimulus of sex pills growth
Resistance plasmid - Contain the genes responsible for encoding for the proteins that allow for antibiotic resistance
Col plasmids - Contain the genes that code for bacteriocins, proteins that can kill other bacteria.
Degradative plasmids - Contain genes for products of enzymes that can break down foreign material
Virulence plasmid - Able to turn the host bacteria into a pathogen |
|
|
Term
| What are the selectable markers in plasmid engineering? |
|
Definition
| These are the antibiotic resistance plasmids that we can use to check whether successful transformation has occurred |
|
|
Term
| What does the pBR322 plasmid contain? |
|
Definition
TETr gene
AMPr gene
ori and rop sites
4361bp |
|
|
Term
| Where are the restriction enzyme sites in the first plasmid, pBR322? |
|
Definition
EcoRI is in the middle of normal plasmid DNA
PstI is situated in the middle of the AMPr gene
BamHI is situated in the middle of the TETr gene |
|
|
Term
| What are the pUC plasmids? |
|
Definition
These were the plasmids discovered by (Viera & Messing, 1982) after the pBR322 plasmid
They have multiple cloning sites for various genes.
Contains AMPr and lacZ
The lacZ is the string of DNA that contains very many sites for all manor of different restriction sites including PstI, EcoRI, BamHI, SacI, SmaI and more
Also allows for alpha complementation |
|
|
Term
| What is alpha complementation? |
|
Definition
This is based around the lacZ gene.
It encodes for an alpha-peptide that will interact with a carboxyl terminus of beta-galactosidase to produce full beta-galactosidase. This enzyme product will cleave a colourless substrate, called X-Gal, giving the products, galactose and 5-bromo-4-chloro-indoxyl.
The indoxyl product will dimerise and then oxidise into a insoluble blue dye.
This is the premise for the white/blue screening
Alpha complementation is the idea that a mutant lacZ gene can be made functional by co-expressing the lacZ alpha-peptide
Means you can take an inactive form of the lacZ gene and then activate it to your control |
|
|
Term
| What is Blue/White selection? |
|
Definition
This is the premise that an untransformed pUC18 vector will cause the release of blue dye in the presence of ampicillin and X-Gal as the X-Gal is successfully cleaved.
As you are inputting a gene into a site in the lacZ gene, successful transformation will disrupt the beta-galactosidase production and then the X-gal will not be cleaved and it will remain colourless.
Blue = unsuccessful transformation
White = successful recombination |
|
|
Term
| What is the name of the mutant lacZ gene in pUC18? |
|
Definition
|
|
Term
|
Definition
It is a bacteriophage vector.
Developed as it could package the DNA as single stranded unlike plasmid vectors
Also allowed for purification of the virus particles leading to purification of the ssDNA
It is encased in a protein coat and once it has been injected into the bacterium (E. coli) it will go into a replicative form (dsDNA)
Once in the bacterium it will replicate 1000s of times and then form 1000s more phages which will lyse the bacteria and continue |
|
|
Term
| What is an M13mp18 vector? |
|
Definition
| This is the same as an M13 bacteriophage vector but it has a lacZ gene input into it so it can be tested for successful recombination |
|
|
Term
|
Definition
This is a plasmid in a phage
Examples are the M13 and the M13mp18
They are:
- Harder to prepare and store however they do contain both an ori site AND M13 f1 origin site. This means it can act as a normal plasmid as well as phagemid. |
|
|
Term
|
Definition
It is the most common phagemid in use today
It contains a lacZ gene, ColE1 ori site, AMPr and the phagemid f1 ori site.
Produced by Stratagene |
|
|
Term
| Wha is Cre site specific recombinase? |
|
Definition
It is a tyrosine derived enzyme that is used to aid the shuffling of one particular target gene from one plasmid into another.
It mediates the recombination with the DNA sequences at specific locations if there are the correct loxP sites present. They must be complementary loxP sites also.
[image] |
|
|
Term
|
Definition
| These are a bacteriophage that is able to infect E. coli |
|
|
Term
| What is the difference between the lysogenic and lytic pathway? |
|
Definition
These are performed by phages, mainly the lambda phages.
Lytic = enters, reproduces and then lyses the cell
Lysogenic = the phage DNA enters the bacterial DNA and is then continuously reproduced as part of the bacterial DNA |
|
|
Term
| How is the lysogenic and lytic pathway controlled? |
|
Definition
There is a small gene in the lambda phage DNA called cII.
When cII is active, the phage will go into the lysogenic pathway
When inactive, it will go into the lytic pathway
It does this by activating cI which will repress the N and cro and therefore stop the lytic pathway and go ahead with the lysogenic pathway |
|
|
Term
| What is the difference between the lysogenic and lytic pathway? |
|
Definition
These are performed by phages, mainly the lambda phages.
Lytic = enters, reproduces and then lyses the cell
Lysogenic = the phage DNA enters the bacterial DNA and is then continuously reproduced as part of the bacterial DNA |
|
|
Term
| How is the lysogenic and lytic pathway controlled? |
|
Definition
There is a small gene in the lambda phage DNA called cII.
When cII is active, the phage will go into the lysogenic pathway
When inactive, it will go into the lytic pathway
It does this by activating cI which will repress the N and cro and therefore stop the lytic pathway and go ahead with the lysogenic pathway |
|
|
Term
|
Definition
| This is the area on a bacterial culture which is clear of growth because phage lysis has occurred as the cII gene has been inactivated in that area and bacteria has lysed. |
|
|
Term
| How many DNA fragments can be packaged into the Phage head? |
|
Definition
37 to 52kb of DNA fragment
23kb of DNA can be placed in vitro into the phage as the middle of the phage genome can be replaced if the lytic cycle is being used.
This means scientists can manipulate 23kb of DNA in a phage. 7kb of that can cDNA |
|
|
Term
| What part of the phage DNA is not required for lytic growth? |
|
Definition
|
|
Term
| How do you package phage particles in vitro? |
|
Definition
By getting two lambda lysogen and taking phage tails and phage heads and mixing them with concatemerized lambda DNA and you get a mature lambda phage with your desired phage particles
[image] |
|
|
Term
|
Definition
| it is a hybrid between a lambda vector and plasmid They can contain 33 to 45kb of material It is very much like a plasmid but contains COS sites which are the only mechanisms required, if you ligate COS sites around 50kb apart, anything that size can be placed in All around the COS site is lambda DNA Can usually be cut with SmaI because it creates blunt ends You can then package these into phage heads |
|
|
Term
|
Definition
|
|
Term
|
Definition
| Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction |
|
|
Term
| What are the temperatures required for PCR? |
|
Definition
| 94ºC - To break the hydrogen bonds 55-65ºC - To allow for the development of new nucleotide chains to be built from primers 72ºC - Annealation of the new strand to the template strand |
|
|
Term
| How many DNA strands does PCR make after 30 cycles? |
|
Definition
|
|
Term
|
Definition
This is the thermo stable polymerase that is used for the PCR reaction, T. aqauticus is found in hot springs naturally
Half-life = 1.6 hours
Will make mistakes as it does not have the 3'-5' exonuclease proofreading tool.
Proofreading polymerases do exist however, examples are:
Pfu Pol (DNA), Tol Pol (DNA) and Tli Pol (DNA)
All these three will give blunt ends however. The Taq Pol will add on a template independent A residue (3 adenosine nucleotides on the end) |
|
|
Term
|
Definition
| In higher concentrations to a optimum level of 4mM, the magnesium aids the PCR reaction in being more specific, creating perfect replicates of the DNA and lessening the need for the 3'-5' exonuclease proofreading mechanism |
|
|
Term
| What should you look for in a primer? |
|
Definition
17-30nts long
Should be about 50-50 GC and AT
Should avoid any long runs of single nucleotides
Annealing temp ~ {2(AT)+4(GC)}
Avoid any specific sequences that may produce an issue (like a hairpin loop like sequence) and not two identical, not to make any primer dimers
Introduce primers that will be complementary to specific restriction enzymes. Introducing purposeful restriction sites |
|
|
Term
| When can PCR be used for other analysis? |
|
Definition
| You can use known primers that will be complementary to the healthy or known mutant DNA sequence and you can run with PCR and then run an electrophoresis. This is to find whether or not the PCR amplification is successful and therefore whether or not a mutation is present or not. |
|
|
Term
| What is a mutation that you can find when analysing with PCR? |
|
Definition
Waardenburg syndrome - find a deletion
Sickle cell anaemia - find a point mutation |
|
|
Term
| How do you add whole PCR products into vectors? |
|
Definition
| You cut the vector using blunt end restriction enzymes such as SmaI and then add 3 Thymine nucleotides on the end to be complementary to the 3As that the Taq Pol adds to the PCR product. 3Ts are added with dTTP |
|
|
Term
| How do amplify a piece of RNA? |
|
Definition
Take the RNA, treat with reverse transcriptase to get a complementary strand and then put through the PCR reaction. This will then, then can treat with RNA polymerase to get a the RNA product.
Not sure how to then isolate the RNA again though |
|
|
Term
|
Definition
This is the use of TaqMan probes to see how much amplification is being undergone in a certain PCR reaction. TaqMan are oligonucleotides contain fluorescent capable probes that when split will glow. There will be two tags in the oligo nucleotide but only one will stay in the DNA sequence. The other will split off when the other is bound to and glow. The fluorescence increases proportionally to the rate of PCR in the reaction.
[image] |
|
|
Term
| What are the key features of a YAC? |
|
Definition
- Autonomously replicating sequence (ARS)
- A yeast centromere
- A yeast telomere
- Genes for YAC selection (URA3 (Uracil biosynthesis) and TRP1 (tryptophan biosynthesis))
- A suppressor tRNA gene (SUP4)
- Bacterial replication origin site (ori) and a selectable marker gene (e.g. AMPr) |
|
|
Term
| How do you engineer a YAC to be a functional piece of engineered DNA? |
|
Definition
First you must know that you can use a ade2-ochre mutant that contains a adenine biosynthesis pathway that gives yeast a red colour and a SUP4 gene that suppresses the adenosine pathway and prevents the red colour from accumulating so it's white instead
Cut with BamHI and EcoRI. BamHI will cut at the telomeres and allow create linear DNA. EcoRI will cut in the SUP4 site which. This stops the SUP4 gene function and a red colour is given off rather than a white colour. This means that when successful transformation has occurred and a gene has be placed into the EcoRI cut site, you can select of red colonies as these are the successfully transformed YACs |
|
|
Term
| What is the YAC selection process for successfully transformed YACs? |
|
Definition
| Transform ade-, ura-, trp- yeast and select for red, URA+, TRP+ colonies. |
|
|
Term
| What happens if you try to clone too large a piece of DNA with a YAC? |
|
Definition
YACs will be able to clone DNA from 200~2000kb.
However when they start getting closer to the 2000kb they are prone to breaking and causing unstable clones |
|
|
Term
|
Definition
| Based on the P1 bacteriophage, we are able to repress the lytic pathway and then the P1 plasmid is used for low copies and only up to 150kb. |
|
|
Term
| What are the main points of the make up of the P1 plasmid? |
|
Definition
- It contains two replication origin sites.
One controls the lytic DNA replication (P1 lytic replicon)
One controls the plasmid replication (P1 plasmid replicon
- pac site: this is cleaved prior to insertion of the phage DNA into the phage particles
- sacB site: this gene is responsible for encoding levansucrase which is toxic for E. coli cells growing on sucrose, it contains the BamHI cut site so acts as a selectable marker for E. coli cells |
|
|
Term
| What enzymes are used to cut the P1 plasmid? |
|
Definition
BamHI which cuts the sacB site
SacI which cuts in-between the ori and AMPr site. It is a blunt end restriction enzyme |
|
|
Term
| How do you package P1 plasmids with desired DNA and then put into E. coli for replication? |
|
Definition
Cut the sacB with BamHI and then insert a piece of genomic DNA (70-95kbp) and ligate.
Then transfect (method of entering the engineered DNA into the E. coli cell) the engineered DNA with Cre recombinase which will also cut the plasmid at the loxP sites so you lose the ori, pac and AMPr sites.
Then this DNA will begin to replicate with the E. coli cells |
|
|
Term
| What are the key features of a BAC? |
|
Definition
It is an engineered version of the F plasmid. This means it contains the Fertility factor oriS which allows it to integrate itself into the bacterial chromosome.
F-factor requires certain gene for maintenance of function:
repE, parA, parB, parC |
|
|
Term
| What is the main issue with BAC? |
|
Definition
It has a very low DNA yield.
You don't get much for what you put in but it is fairly self contained and fairly accurate |
|
|
Term
|
Definition
These are genomic libraries that contain DNA copies of mRNA.
They require reverse transcriptase for creation |
|
|
Term
| How to create a genomic library? |
|
Definition
| Use partial digest so you are replicating many small pieces of DNA that all have obvious overlaps in sequence so you are able to tell where the DNA fragment was located once you have replicated the fragment enough to sequence it. |
|
|
Term
| What are the basics for genomic library construction? |
|
Definition
Cut the piece of DNA you want to replicate and sequence, around 20kb in length.
Treat with EcoRI methylase (This blocks the EcoRI cutting sites on your desired DNA so it is not cut)
Add Linkers (extra DNA for restriction enzyme site) then cut with EcoRI and mix into the plasmid
The put the plasmid containing the desired DNA into a plasmid or whatever and then replicate and remove |
|
|
Term
| How do you know how many independent clones you will need to ensure entire genome is represented? |
|
Definition
It depends on the size of genome and average length of cloned fragment.
(Size of genome)/(Average fragment length) = minimum number you require.
You in fact require many many more than this to make sure you have enough.
Must be about 6.25x more cDNA than the minimum amount |
|
|
Term
| How to make a cDNA library? |
|
Definition
Must take purifies mRNA, add a oligo-dT primer onto it and then use revers transcriptase to create the cDNA strand.
Then using terminal transferase, dCTP and TdT (terminal deoxynucleotide transferase)
TdT is used to catalyse the addition of nucleotides on to the 3' end of the cDNA
Then using an alkaline sucrose gradient you can split the cDNA from the mRNA.
Then addition of another oligo-dG to pair with the poly-C tail made by the dCTP. Then more reverse transcriptase to create ds cDNA. |
|
|
Term
|
Definition
This is a primer that will attach the poly-A tail or a poly-C tail.
Therefore oligo-dG is form attaching to poly-C tail and all the other variants. |
|
|
Term
| What is important about the orientation of the cDNA in a cDNA cloning? |
|
Definition
The direction the cDNA is facing must be correct, it is always 5'-3' direction away from the promoter in the vector that you will be adding the cDNA to for cloning.
This means you must orientate it specifically using different restriction site sensitive primers on the correct ends of the cDNA. |
|
|
Term
| What is the subtraction cDNA library technique? |
|
Definition
This is for the purification of a cDNA library. It removes the sequences in a cDNA library that are common between two separae libraries allowing for more cell or tissue specific cDNA libraries
Do this by using labelled driver cDNA (labelled with Biotin) which will bind to the complementary cDNA from the tester cDNA an this means that you can put it through and Avidin column so the biotin marked cDNA will be stopped so you can collect the cDNA from the tester cDNA sample that is unique.
You can then enrich the gene that was unique to the tester cDNA and then you have a large amount of cDNA that is unique to that particular library |
|
|
Term
| How do you screen for genes in DNA with nitrocellulose and x-ray? |
|
Definition
This is a selection process to find a successfully replicated DNA plasmid
Place nitrocellulose paper onto petri dish of recombinant bacteria colonies that may contain the plasmid you're looking for. Peel off paper and lyse bacteria and denature the DNA.
incubate with a known radioactively labelled probe and then expose to x ray and then it will show the bacterial colony that contains the recombinant plasmid you were looking for. Then you can compare this to your petri dish to have the right colony with the correct plasmid |
|
|
Term
| What is immuno-screening of lambda phage libraries? |
|
Definition
It is a selection process to find a successfully replicated DNA plasmid
This is when you plate the lambda phage library onto a bacterial lawn and then use nitrocellulose paper to take a copy of petri dish colonies.
Then you can use known radioactively labelled antibodies to attach to the positively recombinant plasmid that has successfully replicated in a bacterial colony |
|
|
Term
| What is a two hybrid screen? |
|
Definition
It is the mechanism by which a bait protein is used that is bound to a DBD and a prey protein which is bound to a Transcriptional activation domain.
This means that if the prey is present in certain sample, the bait and prey will bind, which will in turn bind the DBD and TAD together an they will bind to a certain reporter gene promoter site and till begin to transcribe a certain reporter gene to indicate that the prey protein is present.
TAD may just be known as Ad (activation domain) |
|
|
Term
| What is the key to creating the makeup of the two hybrid system? |
|
Definition
The prey and the bay proteins must come from two specific plasmids that will contain known cDNA in them. This means you can screen for the particular cDNA plasmid by knowing what prey protein to look for.
In MGaG case, we use the Gal4 plasmid. |
|
|
Term
| What result will you get if you use LacZ as a reporter gene? |
|
Definition
| Positive prey-bait interaction will result in a blue colony due to successful LacZ gene transcription. |
|
|
Term
| Apart from the two hybrid screening process, which other hybrid screening processes are there? |
|
Definition
One hybrid for DBD
Three hybrid for RNA binding proteins - Uses RNA binding proteins attached the Gal4 DBD and Gal4 TAD instead and a bifunctional piece of RNA
Three hybrid for ligand-binding proteins - Uses Ligad binding proteins attached to the Gal4p Ad and the LexA DBD instead and a bifunctional ligand. |
|
|
Term
| What are the two kinds of cell lines that we can create? |
|
Definition
Continuous cell line - creating a cell line that will last forever and we can always take from it
Transformed cell line - a cell line that will contain transformed cells |
|
|
Term
| What is the difference between Normal cells and Transformed cells for transformed cell lines? |
|
Definition
Normal cells:
- finite lifespan
- altered characteristics as the cell ages
- require solid anchorage
- will stop replicating at certain cell density
Transformed cells:
- continuously culturable
- infinite lifespan
- quicker population doubling time
- independent of solid anchorage, can multiply in liquid medium
- genetic instability (can have random numbers of chromosomes)
- altered growth control (can control growth more easily and can replicate as many times as required, independent of density)
- fairly self dependent |
|
|
Term
| What are the famous cell lines? |
|
Definition
|
|
Term
| What are expression vectors and what are they used for? |
|
Definition
| They are vectors that are used to either control gene expression or control different factors in DNA. They are used to study the different gene factors and activity. |
|
|
Term
| What are the four different kind of expression vectors? |
|
Definition
Expression vectors will contain different genes and mechanisms to study different areas of gene function.
Minimal promoters used to study gene regulatory elements such as enhancer elements.
Constitutive promoters used to direct expression of gene products.
Cell-specific promoters used to specify expression to target cells.
Regulated promoters used to control the on/off expression of cloned genes. |
|
|
Term
|
Definition
This is the process by which you are putting the desired genes inside the mammalian cell.
It is the mammalian version of transformation and there are many methods.
It was first described by (Szybalska & Szybalski, 1962) |
|
|
Term
| What is the most common selectable marker gene for uptake of gene by mammalian expression vector? |
|
Definition
Neomycin.
Others include hygromycin transferase, puromycin acetyl transferase, bleomycin binding protein and xanthine-guanine phosribtransferase |
|
|
Term
| What are the methods of transfection? |
|
Definition
- Gene gun
- Micro injection
- Liposome-mediated
- electroporation
- retroviral
- DNA-calcium phosphate co-precipitation |
|
|
Term
| Explain the DNA-calcium phosphate co-precipitation use for transfection |
|
Definition
| You wash the cells with a phosphate buffer and then add the CaCl2 and the DNA to the cell growth medium. The DNA is precipitated by the calcium chloride and then the DNA just enters the cell through endocytosis |
|
|
Term
| Explain the liposome mediated method of transfection |
|
Definition
| Mix polycationic lipids and neutral lipids to create unilamellar liposome vesicle (liposomes with a surrounding positive charge). This allows the DNA to be packaged inside a liposome complex and then the complex is taken in by the cell by endocytosis |
|
|
Term
| Explain the electroporation method of transfection |
|
Definition
By manipulating the strength and duration of electric field that a mammalian cell is kept it, you can create differing size pores in the cell membrane and then you can make small enough pores that will be able to repair themselves when the DNA has entered.
This method kills a lot of cells and therefore it is not too efficient by it is the most versatile method |
|
|
Term
| Explain the nucleofection method of transfection |
|
Definition
It is the same as electroporation where you create pores with electricity however it can ensure direct delivery in the nucleus which means that expression will occur around 4 hours after transfection.
Also means that the DNA in the cytoplasm will enter the nucleus upon cell division which means that the transfection efficiency increase over time |
|
|
Term
| How can you use retroviruses for transfection? |
|
Definition
You can manipulate a retrovirus to carry the desired expression DNA that you want as your expression vector.
It will infect 100% of the cells in question which makes it very efficient but it is costly and very hard |
|
|
Term
| What are the components of a retrovirus? |
|
Definition
Linear DNA with a an LTR at either end which will contain the enhancer/promoter and PolyA.
And then genes that will encode the following:
ψ: packaging site
gag: capsid proteins
pol: reverse transcriptase
env: surface and transmembrane proteins. |
|
|
Term
| What are the main issue and goal when using retroviruses as expression vectors? |
|
Definition
| You must be able to control the retrovirus as they can be very contagious and dangerous The goal with a retroviral vector is to be able to use the retrovirus to get the DNA into the mammalian cell but you do not want the retrovirus to replicate. You do this by separating the ψ: packaging site from the desired genetic material You are able to remove the gag, pol and env sites and then just have these in the retroviral espression vector and dispose of the ψ: packaging site. These components are then put into the packaging cell lines (without the ψ: packaging site which is confusing) |
|
|
Term
| Explain the basic method of creating a retroviral vector |
|
Definition
Create a packaging cell line first. This is a cell that will contain the gag, pol and env sites and then just have these in the retroviral espression vector and dispose of the ψ: packaging site.
Then you mix the packaging cell line with the packaging positive retroviral vector. The retroviral vector then enters the packaging cell line and creates a retroviral vector that contains the correct expression genes, the reverse transcriptase genes, capsid protein genes, surface membrane genes but NOT the packaging genes and therefore the retrovirus is unable to replicate once it has been administered to the target expression cell
This means you can control it |
|
|
Term
| What is an Aden-associated virus? |
|
Definition
It is a virus that contains a single stranded DNA genome. It looks similar to the retrovirus DNA but instead of LTRs on either end it has ITRs (Inverted Terminal Repeats) and they created hairpin structures on themselves.
[image]
It can infect dividing and non dividing cells, infect wide variety of cell including neurons
It will infect humans at a specific locus on chromosome 19!
Can be used by replacing the genes with the desired foreign DNA and then mixing it with the desired cells. The rest will be done in vivo |
|
|
Term
| How do you use a AVV as a vector? |
|
Definition
| Remove the genes that are usually present between the ITRs and then replace with a transgene expression cassette, CMV (internal promoter) and a PolyA sequence. Then just mix with the target cells and it will be done in vivo |
|
|
Term
| How do you make the recombinant AAV vectors? |
|
Definition
Mix viral proteins with a piece of foreign DNA containing an ITR on either end.
Mix into a cell line of 293 cells (embryonic kidney cells that are good for transfection).
These will the produce the sDNA contain the ITRs and the foreign gene.
Then mix that with more viral proteins and the virus will be formed and then can be release to infect target cells |
|
|
Term
| What is the idea of transient transfection? |
|
Definition
This is the idea that the DNA taken up by mammalian cells will only last a short while before being degraded by the host cell.
However this 24-48hr transient period is long enough for analysis of the gene expression and protein function in the target cell |
|
|
Term
| What are the average efficiencies of the different mammalian expression vector methods |
|
Definition
30-40% - electroporation and lipofection
60-80% - nucleofection
Higher than all - viral use (retrovirus and AVV) |
|
|
Term
| What is the key to a stable cell line? |
|
Definition
Introduce a recombinant vector in low frequency to a host cell and that will integrate itself into the host cell genome.
Because it integrates into the host chromosome, it results in stable replication of the foreign gene.
You can then be able to select more specific cell lines by additional expression of resistance genes so that you can get specific cell lines from a more general cell line |
|
|
Term
| What is the use of cell lines that contain foreign genes? |
|
Definition
cell cycle control
signal transduction
programmed cell death
oncogenesis
transcriptional control and gene regulation protein interactions
identification of domain function |
|
|
Term
|
Definition
It is the Green fluorescence protein, it is 238AA long and originally from A. victoria jellyfish (Prasher et al., 1992)
It works by absorbing light emitted by another protein called aequorin of a short wavelength and then the GFP will fluoresce at a longer wavelength (nearer the red end at the green area)
The process is calcium dependent.
It was found however that the GFP would fluoresce without any aequorin or calcium if it was excited by UV light |
|
|
Term
| What is the molecular structure of GFP? |
|
Definition
| Beta-barrel comprised of 11 beta sheets surrounding a central cyclic tripeptide called chromophore. |
|
|
Term
| What is vector we use in animals for the introduction of the GFP protein? |
|
Definition
|
|
Term
| How can introduce the GFP protein into animal cells? |
|
Definition
| Taking the pEGFP-C1 vector contain the gene for GFP production and then adding a gene into the MCS to allow for expression. Commonly fused with a cytoskeleton gene so that the development of the cytoskeleton includes GFP so the cytoskeleton goos in the dark. |
|
|
Term
|
Definition
Fluorescence loss in photobeaching
It is a technique to understand the stability of certain proteins by looking at the amount of fluorescence still present after numerous round of photobleaching. The longer the fluorescence sticks around, the more stable to protein |
|
|
Term
|
Definition
| This is a protein that is a guanine exchange factor that activates Rho GTPases. |
|
|
Term
| What did (Jaenisch & Mintz, 1974) find out about animal transgenics? |
|
Definition
They found that purified SV40 DNA could be injected into the blastocoel cavity of an embryonic mouse and this would then integrate into the embryos genome.
In 1976, Jaenisch found that you could safely implant a retrovirus (the Moloney murine leukaemia virus specifically) by viral infection of the preimplantation mouse embryos. |
|
|
Term
| What did (Anderson et al.;Capecchi, 1980) find out about animal transgenics? |
|
Definition
| They found that is you took purified DNA and microinjected it directly in fibroblast cells, the DNA would stable incorporate itself into the cells genome and would be expressed |
|
|
Term
| What did (Gordon et al., 1980) find out about animal transgenics? |
|
Definition
| They were the first people to be able to integrate a cloned gene into a somatic cell using pronuclear injection |
|
|
Term
| What is pronuclear injection? |
|
Definition
This is the injection of DNA into the nucleus of one of the nuclei present in the fertilised egg prior to the fusion of the male and female nuclei to form the complete zygote
Injection usually into the male nuclei as its bigger
Done by using a small holding pipette and a tiny needle |
|
|
Term
| What is pronuclear injection used for? |
|
Definition
This is the gain of function transgenics.
It is used to give an animal a certain gene or set of genes in their pre zygote stage so that they are born with additional desired function |
|
|
Term
| How do you test whether pronuclear injection transgenics has been successful? |
|
Definition
| Isolate DNA from the transformed animal and then treat DNA with PCR and then run electrophoresis against control |
|
|
Term
| How efficient is pronucleus injection transgenics? |
|
Definition
| 10-40% of cells will contain the desired integrated foreign DNA |
|
|
Term
| What are the potential issues with pronuclear injection? |
|
Definition
Random integration anywhere in the genome
Can be copied multiple times into the genome
1/10 chance it will cause an insert mutation |
|
|
Term
| What does cis-acting mean? |
|
Definition
This is when a molecule will be on the same molecule of DNA as the gene that it is controlling or the mechanism it is part of.
A promoter region is a cis-acting mechanism |
|
|
Term
| What does trans-acting mean? |
|
Definition
This is when a molecule is not part of the certain gene or complex that it is acting on. May be a free molecule that will act on a certain area of DNA and control it.
Easier to control trans-acting element than cis-acting elements as you can control the concentration of them for example |
|
|
Term
| What is the idea of 'knockout mice'? |
|
Definition
This is the idea that you are bale to stop a function of a developing mouse, prevent some genes from being expressed.
Requires embryonic stem cells and recombination via the Cre-loxP system. |
|
|
Term
| When were embryonic stem cells first derived? |
|
Definition
In 1981 by (Evans & Kauffman) and (Martin)
Get them from the blastocysts of embryonic mice. This is about 3.5 days after the zygote has formed. |
|
|
Term
| How do you get large numbers of embryonic stem cells? |
|
Definition
| Take the stem cells from the blastocyst of embryonic mouse and then treat with Leukaemia Inhibition Factor (LIF) and this causes mass replication. |
|
|
Term
|
Definition
| It is the idea of an organism with a mix of genes from two different zygotes. Taking the genes from on zygote and placing them into another to see how the mixture of two genotypes fairs. |
|
|
Term
| How do introduce desired DNA into a germline? |
|
Definition
Manipulate embryonic stem cells to include desired DNA
microinject these stem cells into another blastocyst and allow to develop into an adult mouse. This mouse is a chimeric mouse as it has the genes from two different genotypes.
Then breed chimeric mouse with WT and then the DNA will be fully integrated into the germline of the mouse |
|
|
Term
| When was the first use of genetic manipulation of stem cells? |
|
Definition
Robertson and Gossler (1986)
They demonstrated that a retrovirus or neomycin could be transmitted through the germline (nothing was specific though)
Hooper and Kuehn (1987) then targeted specific HPRT gene by homologous recombination |
|
|
Term
| How was the first genetic manipulation of ESC done? |
|
Definition
By taking a gene vectors and then causing homologous recombination with the stem cell DNA.
You use integrate selectable marker to recriminate also so that you are able to select the successfully recombined cells.
Then you can introduce these stem cells into a WT blastocyst of a mouse and then create a chimeric mouse. Then just raise and breed with WT mouse to get the desired DNA into the germline |
|
|
Term
| What are two selectable markers used in genetic manipulation of ESC? |
|
Definition
Neomycin gene
HSVk gene
Neo selected by treating with G418 to remove cells WITHOUT it
HSVtk selected with by treating with Ganciclovir to remove cells WITH HSVtk |
|
|
Term
| What are some issue seen creating knockout mice that genetically manipulated ESC in their developing blastocyst? |
|
Definition
Developmental defects
Die as embryos
Gene may only be cell lineage specific and therefore either hard to see or destroys entire cell lineage of certain aspect |
|
|
Term
| Can you use LacZ in the process of gene knockout in the manipulation of ESC? |
|
Definition
| Yes, LacZ is often used as it is easy to use and select for |
|
|
Term
| What is the Cre-loxP system? |
|
Definition
It is a site specific recombinase that will be able to remove the gene that is located in-between two loxP sites in a sequence.
Cre is the recombinase that recognises the loxP sites.
Cre is a 38kDa from bacteriophage P1
loxP site is a 34 bp long |
|
|
Term
| What are the two ways Cre can act on the loxP site in recombination? |
|
Definition
It can either invert the middle section of DNA and leave the two loxP sites or can delete the middle DNA part along with one of the loxP sites
This means that sections of DNA between two loxP sites can easily be removed |
|
|
Term
| Where can Cre-loxP recombination be done? |
|
Definition
In ESC or in the organism itself
Cre can be very tissue specific and therefore you are able to manipulate the Cre to become a tissue-specific Cre transgene which will only recombinase the tissue in question.
This overcomes the problem with basic gene knockout ESC manipulation where the cell lineage may have been specific |
|
|
Term
| How can you create inducible Cre activity that you can control when to turn or or not? (in regard to Cre-loxP gene manipulation) |
|
Definition
Can add HSP90 (heat shock proteins) that will gather around the Hb-Cre-Hb complex that you can use by ligand binding.
This means the complex is too large to enter the nucleus where the DNA with the loxP site is.
You can then add a molecule called TAMOFLEXIN which will breakdown the complex and Cre can enter the nucleus and recombination can occur
Tamoflexin is administered in drinking water of the organisms as it is a hormone |
|
|
Term
| What is the tetracycline operon? |
|
Definition
Made up of:
tetO: tetracycline responsive operator
tetR: tetracycline repressor
tc: tetracycline (what binds to the repressor to allow for transcription)
The transcribed genes can be changed and is predominantly used to control Cre production. |
|
|
Term
How can a herpes simplex virus (VP16) be used to control the tetracycline operon and Cre production?
Also called the tTA system |
|
Definition
The VP16 molecule will bind to the tetR-tetO complex and cause activation of the TATA box which acts as a minimal promoter for the Cre. So only small amount of Cre are made
In this case, with present VP16 and the TATA box, the addition of the Tc will STOP transcription rather than promote it.
This is known as the tTA system |
|
|
Term
| What is the rtTA system in Cre production control with a Tetracycline operon? |
|
Definition
| It is the reverse of the standard system whereby mutant tetracycline repressor will bind to the operator when both tetracycline and the VP16 molecules are present and therefore the TATA box begins low level CRE expression |
|
|
Term
| How can the tTA system and the rtTA system be altered to be more specific? |
|
Definition
By adding a tissue specific promoter to the promoter end of the operon, this will then have to activated by tissue specific proteins. This means that Cre production will only occur in that specific tissue.
Therefore you can analyse gene function by knockout in specific tissues |
|
|
Term
| What is the most famous method of DNA sequencing? |
|
Definition
Sanger method
This is the process by which you add tagged dideoxynucleotides (these will terminate the sequence as cannot be bound to on the 3' end) so that you are able to add them and then analyse through through laser detection what the nucleotide is at the different points in the sequence. Get a graph like this...
[image] |
|
|
Term
| How did you obtain ssDNA in the early methods of DNA sequencing? |
|
Definition
Add DNA into a M13 or phagemid vector and transfer the vector into E. coli.
replication will occur and ssDNA will be produced
This was then used as the template DNA for sequencing. |
|
|
Term
| What is the Klenow polymerase? |
|
Definition
This is the larger fragment proteolysis product when you break down the DNA polymerase I holoenzyme.
It is made up of the polymerase domain and the 3'-5; exonuclease domain (it looses the 5'-3' exonuclease domain).
Cleaved by subtilisin enzyme.
Klenow polymerase is heat sensitive and will become inactive after 20min at 75ºC |
|
|
Term
| What is special about the primer used in DNA sequencing? |
|
Definition
They are about 20bp in length comprised of synthetic DNA
In fact very similar to the regular primers |
|
|
Term
| What are the dideoxynucleotides and why are they so special? |
|
Definition
These are used in DNA sequencing as you can mix a few radioactively labelled ddNTPs into the mix and they will bind and because they have no OH group on the 2' or 3' carbon, they are unable to create a phosphodiester bond and therefore the strand after the ddNTP is terminated.
These are then the bases that are analysed by either polyacrimide electrophoresis and x ray film or laser to determine the location of each ddNTP in the sequence and analyse the sequence |
|
|
Term
| What was the early way to analyse the sequence of DNA once you had added the radioactively labelled DNA and the primers and such? |
|
Definition
With polyacrimide gel electrophoresis. It is a very accurate electrophoresis that will split the DNA strands that are one nucleotide longer or shorter and then scan with x-ray film to see what the ddNTP in the sequence is.
Long process for millions of NTPs |
|
|
Term
| What is cycle sequencing? |
|
Definition
Sequencing method performed with dsDNA
Used only one primer
Similar to PCR amplification
Uses ddNTPs
Uses Taq polymerase instead of Klenow pol as it allows for a more efficient incorporation of the fluorescent ddNTPS
Uses the 95ºC to 55ºC to 72ºC like in PCR to incorporate the ddNTP in all of the sequence points
Then you use the polyacrimide gel electrophoresis again |
|
|
Term
| What is automated Sanger sequencing? |
|
Definition
This is the idea that you still use the ddNTPs at different points in the sequence however you use fluorescent markers instead of radioactive and use scanners instead of x-ray film.
Thats when you get the scan that looks like this:
[image] |
|
|
Term
| What were the first two improved methods to the automated Sanger sequencing method that were used to increase productivity? |
|
Definition
Automated gel sequencing: Allows 36 samples of 450bp of sequence in 24hrs. So 150fb per 24hrs
Capillary sequencing: uses small capillary tubes rather than gel plates which means smaller space is used. Can do 950kb per 24hrs |
|
|
Term
|
Definition
| It is a newer method of sequencing the sequenced james Watson's genome in 4.5 months and $1.5 million. Original human genome project took 13years and $2.7 billion |
|
|
Term
| What is pyrophosphate sequencing? |
|
Definition
| This is the process you add the 4 dNTPs 1 at a time to a building sequence and then whichever dNTP binds to the complementing strand. When a complementing base actually does bind, the two phosphate molecules that are remove in the phosphodiester bond creation react with sulfurylase in the medium to create ATP and then the ATP will excite the luciferase in the system which then causes light emission which is detected by a scanner which will then record which base it was that caused light emission and therefore which base it is in the sequence. |
|
|
Term
|
Definition
It is the enzyme used in DNA sequencing that can emit light when phosphate is broken down by sulfurylase and then the ATP excites it.
This is in the pyrophosphate sequencing reaction |
|
|
Term
| What is massively parallel signature sequencing? |
|
Definition
This is the micro bead process.
It breaks down the genome into small different fragments and then attaches the different fragments to individual microbeads.
Microbeads are encased in oily PCR stimulating substance that causes the fragment to amplify 10 million times
The oil is then washed off and the DNA is denatured leaving ssDNA
Each bead with the attached ssDNA is then placed into small individual well.
The pyrophosphate method is then used by flushing dCTPs over all the wells and then the wells that emit light will be detected due to sulfurylase and luciferase and scanner detection.
If a string of a certain dNTP is added then a higher emission of light is detected and will correspond to the amount of dNTPs added.
Becomes inaccurate when large strings of PolydNTPs are added.
Able to sequence 25million bps in 4 hours |
|
|
Term
| What can cause inaccuracy in the pyrophosphate sequencing method? |
|
Definition
When large numbers of repeated base are added to the sequence the intensity of the light emission will become hard to decipher how many there exactly are.
Long stretches of PolydNTPs are hard to distinguish and give a value to |
|
|
Term
| What is Ilumina/Solexa sequencing? |
|
Definition
This is a process by which a reversible labelled dNTP terminators are used.
They are added like in the Sanger sequencing and the fluorescent tag will emit light when scanned but we can remove the terminator and the tag so that the we can continue to just add and scan using DNA polymerase
Then the the tag is cleaved off the |
|
|
Term
| What is the key point about Ilumina/Solexa sequencing? |
|
Definition
| It uses REVERSIBLE labelled dNTPs so you can remove the terminator and continue the sequencing overtime a nucleotide is added and scanned |
|
|
Term
| What is Oxford nanopore DNA sequencing? |
|
Definition
This is a process that is able to sequence single pieces of DNA in real time by passing the DNA through the nanopore. The nanopores are on a flow cell, about 512 nanopores to a flow cell.
It is said that the nano pore can sequence DNA at a rate between 40-500bp/second |
|
|
Term
| Who does nanopore sequencing work? |
|
Definition
Nanopores exist all over a synthetic polymer membrane with a electronic gradient so that the DNA will pass through.
Each pore contains an adapter in the pore to detect the base going through. The adapter notice changes in the current when the bases go through. The 4 different bases will make 4 unique changes to the current and therefore identification of the bases is recorded based on the changes in the current. |
|
|
Term
| What are the adapters involved in nanopore sequencing method? |
|
Definition
Adapter in the nanopore itself to stop each base and cause the different reactions in the current to cause detection of the different bases
Adapter to join the 5' and 3' end of the DNA to know when the piece is finished
Adapter on the start end to act as a target for the unwinding progressive enzyme that feeds the DNA through the nanopore |
|
|
Term
| What is the main issue with nanopore sequencing? |
|
Definition
| The accuracy is questioned and also it, like the polyphosphate sequencing method,has trouble detecting homopolymers (long stretches of a repeated base) |
|
|
Term
|
Definition
| This is a long stretch of repeated bases e.g. ACGCCCCCCCCCCCCCCCCCCCCCTGA where the C is a homopolymer |
|
|
Term
| What are the advantages and disadvantages of the nanopore sequencing? |
|
Definition
Ad:
-Portable
- Data seen as it is done, don't need to wait for complete sequencing
- Can generate single sequences for long molecules
Dis:
-Not commercial yet
- High error rate
- High cost
- Under development |
|
|
Term
| What is the basis of gene function study by mutation creation? |
|
Definition
| Site specific ability to change the sequence of specific proteins, regulatory elements thereby either repressing or altering the expression. |
|
|
Term
| How do you create a mutation with the four primer method? |
|
Definition
In 3 PCR reactions, you use four different primers.
Various annealing and allowing of mutation to arise. This is done by the four different primers. Also you require Klenow polymerase to extend the DNA that contains single point mutations. |
|
|
Term
| What is Alanine scanning for? |
|
Definition
| This is a process by which you can analyse protein function by systematically changing an amino acid in the protein's functional domain to an alanine AA. This will cause |
|
|
Term
| Why is Alanine used for the alanine scanning instead of some other replacement AA? |
|
Definition
It is non bulky, contains a methyl functional group, it is chemically inert and will mimic most secondary structure preferences that other AAs will have.
It is like the universal blood group of the A world |
|
|
Term
| What is an enzyme to use to analyse protein function through random sequence mistakes? |
|
Definition
Taq polymerase as this has no 5'-3' exonuclease proof reading function.
Makes about 1 error per 50,000 bases inserted.
Tfl polymerase makes around 1 mistake per 500,000 bases inserted
Pfu polymerase make around 1 mistake per 1,3 million bases inserted
These can be used in PCR to cause random mutagenesis |
|
|
Term
| What are the basic in vivo and in vitro protein techniques for testing protein function? |
|
Definition
In vivo - insert transformed DNA into a cell. Cell will produce mutant protein and then test the protein product
In vitro - Introduce transformed DNA into a cell, let the cell create mutant protein and then remove protein, purify the protein and then test the protein. This is high level regulated expression and is easier to study what is happening |
|
|
Term
| What is the classical promoter system to regulate gene expression in transformed bacterial plasmid? |
|
Definition
|
|
Term
| Where are the regions of attachment for the promoter before attaching to the operator site? |
|
Definition
| The -35 and -10 sites. They are the primary transcriptional start sites. |
|
|
Term
| How is the leaky Lac promoter combatted for tighter gene expression? |
|
Definition
The use of the the DE3 gene complex in the E. coli genome.
The DE3 complex will contain lac promoter and genes for T7 RNAPol.
The T7 RNAPol will then be the promoter binding to the target gene promoter region turn on gene expression.
The leakiness is controlled because T7 RNAPol is inactivated by a T7 lysozyme. T7 is at low expression therefore will only bind to a concentration of T7 RNAPol that is produced when leaky. When actually being expressed, the T7 lysozyme is in too low a conc. to prevent the target gene expression |
|
|
Term
|
Definition
This is the use of the a tag that will have high affinity to a ion such as Histag affinity towards Ni2+.
The Histag will then be used for pulling the correct sequence out as it binds to the nickel.
Then wash nickel-protein complexes with imadazole which will have preferential binding to the nickel and will wash the protein off which gives you purified protein |
|
|
Term
| What is the IMPACT system? |
|
Definition
This is a method for purifying protein.
Target protein will bind to an intein which is bound to a chitin bind domain.
Run this mixture through a column of chitin beads and when the chitin binding domain binds to the beads.
Then wash with DTT buffer and this will cause the intein to cleave itself and the pure protein will come out with the DTT |
|
|
Term
| What are the selectable makers in haploid Yeast cells? |
|
Definition
|
|
Term
| What is the GAL4-GAL complex in gene expression? |
|
Definition
This is a galactose controlled gene system.
It uses a GAL4 activator will will bind to the promoter ONLY WHEN there is galactose present AND there is no glucose present. It will produce a Gal4p protein which will act as a transcription promoter for the target protein expression vectors. Therefore when Gal4p is transcribed the target protein is produced
Can be modified in some yeast strains that will mean that with you can have the GAL4 gene linked to a stronger promoter which leads to much higher GAL4p production. This then leads to a much higher rate of target protein production |
|
|
Term
| What are examples of reporter genes? |
|
Definition
lac Z (Beta galactosidase) – X gal
GUS (Beta glucoronidase) - glucoronides
Chloramphenicol acetyl transferase - chloramphenicol
Luciferase - luciferin
Secreted alkaline phosphatase |
|
|
Term
| How can Northern Blotting be used to measure the amount of gene expression? |
|
Definition
Electrophoresis to separate the RNA and then the x ray film lay over and hybridised onto the x ray film. Then you are able to use radioactively labelled DNA probe to detect the presence of the target RNA.
The more radioactivity = higher expression which is shown by a stronger band on the scan
Has low sensitivity |
|
|
Term
| What is real time quantitative PCR? |
|
Definition
qPCR is the idea of when using RT-PCR to amplify a certain amount of mRNA, you can measure the amount of molecules of DNA you have. Allows you to follow the process in real time and can measure the amount of DNA you have after each cycle so you know when the PRC product is doubling.
This is to be done as usually PCR production doubling rate is not linear all the way though the process. |
|
|
Term
| What is the technical ingredient in real time quantitative PCR? |
|
Definition
Sybr Green
This will only bind to double stranded DNA and will be fluoresce. The amount of fluorescence seen in the PCR reaction will equate to the amount of PCR product that has been produced |
|
|
Term
| What is TaqMan in real time PCR? |
|
Definition
| This is to detect the amount of PCR product that has been made. Similar to the Sybr-Green label. The TaqMan molecule is a DNA molecule that has a complementary sequence to the DNA sequence you are amplifying. Looks like this: [image] The 3' is a quencher The 5' end is the Fluorophore The fluorophore is released when the TaqMan molecule is linked into the DNA sequence and this release will emit fluorescence. The amount of fluorescence will show how many TaqMan molecules have been involved in the DNA strands and therefore how much PCR has occured |
|
|
Term
| What is a CT value in real time PCR? |
|
Definition
| This is when you are analysing how much PCR has occurred. The CT value is the number of cycles it takes for a particular PCR reaction to stop making the multiplying the DNA strand in a linear fashion. Can be used then to find out how much RNA molecules you had at the start o the process. |
|
|
Term
| What is Nuclear run-on assay and how does it work? |
|
Definition
It is a method to measure the amount of ongoing transcription occurring in a cell.
Lyse the cell in buffer and stop cell processes
Add isolated nuclei containing radioactive UTP and unlabelled NTPs which will bind and label all the RNA that are currently under production because the UTP will activate the RNA pol again and then after this, no new RNA production will occur
Means you can assess how much RNA transcription s currently occurring in a certain cell.
Then measure using filter hybridisation which uses known cDNA sequences to bind to the run-on assay product and then in the you can measure the radioactivity in the filter that has the known cDNA sequence |
|
|
Term
| What is a microarray and how do you make one? |
|
Definition
It is used to measure gene expression in large pieces of DNA and genome.
It is made by having high expression of specific genes and then placing amplifying and purifying them and placed on known areas of a glass sheet. The DNA is then denatured to become single stranded and then UV dried into place.
The glass sheet will have very many different cDNA genes on it with all a known position
Can analyse labelled cDNA to see where and how much radioactivity is shown at different genes , thus corresponding to the level o certain gene expression in a certain genome |
|
|
Term
| What is a two channel array? |
|
Definition
This is using a microarray to see the effect of different growth conditions on the expression of different genes.
Take two cells grown in different conditions, take RNA and produce labelled cDNA, both cell's cDNA being labelled differently. Then mix the two cDNAs together and hybridise onto microarray. The different levels of expression on each gene can then be seen corresponding to the different growth conditions |
|
|
Term
|
Definition
| This is the same idea as a microarray but it dries known oligonucleotides onto the chip instead of using denatured cDNA and it uses a silica surface rather than glass |
|
|
Term
| What are the differences between microarrays and gene chips? |
|
Definition
Microarrays are:
Custom made
Cheaper
Easy use
Gene chips are:
More sensitive
More consistent
Easily purchased |
|
|
Term
| What is direct sequencing? |
|
Definition
| This is a RNA sequencing method which takes the mRNA from a genome and then breaks it up and makes into cDNA, then sequences the cDNA and will map each cDNA sequence onto the genome and then measure the number of reads per gene to measure expression |
|
|
Term
| What are the advantages to RNA-Seq compared to microarrays/genechips? |
|
Definition
Able to measure high and low expression more accurately
Can distinguish between alternatively spliced combinations of the same gene
Can differentiate different alleles of the same gene |
|
|
Term
| What is Chromatin immunoprecipitation? |
|
Definition
ChIP, this is a method used to analyse interaction between proteins and genome
It uses epitope tags, which are known sequences that you can put at the end of a protein sequence and it can then be recognised by labelled antibodies.
It can be for recognition of specific binding sites of regulatory proteins in the genome.
Can also mix this method with the microarray method and create specific microarray which will then have corresponding intergenic regions on it that will bind to the specific antibody labelled pieces of DNA to see how much they are expressed as well
This is the ChIP-chip method |
|
|
Term
| What is the ChIP-Seq method and what is it for? |
|
Definition
| This is using Chromatin Immunoprecipitation method of isolating specific binding sites for regulatory proteins (e.g. TF) and then running them through RNA-Seq to find out how highly expressed the particular regions are |
|
|
Term
| What is PCR-based gene targeting in yeast? |
|
Definition
Functional genomics in yeast
Can manipulate any sequence, some deletion and tagging are also possible
Uses a plasmid which will be inserted into the yeast cell. The plasmid will contain a the target gene called a cassette (e.g. KAN cassette) which will then replace a gene in the yeast DNA by transformation
Must not replace a gene that the yeast requires for survival
The gene you insert will be the gene you are looking at and will first have to amplified in the yeast cell with PCR. Can test successful transformation with antibiotic resistance which the genes will also be in the cassette |
|
|
Term
| What is functional genomics and what are the four systems that are used? |
|
Definition
This is the idea of disrupting gene function to then witness the phenotypic change so you can see what the gene does.
-PCR based gene targeting (yeast)
-Anti-sense morpholinos (animal embryos)
-RNAi (C. elegans, mammalian cell culture)
-The CRISPR-Cas9 system (animals, plants and fungi) |
|
|
Term
|
Definition
This is a synthetic molecule which acts like a piece of single stranded DNA
It can be injected into ANIMAL EMBRYOS and it can be transcribed but it cannot be translated
It means you can engineer a specific morpholine fragment to target specific gene so that all other transcription and translation can occur but the gene that the morpholino is complimentary for, will not be transcribed and will not have functional product
Therefore you can analyse the effect of the loss of that gene product |
|
|
Term
| What is co-suppression/quelling? |
|
Definition
This is used in functional genomics for PLANTS AND FUNGI and it is the suppression of certain genes by adding many copies of the certain genes (transgenes). Just by adding multiple copies of a certain gene it will suppress that gene.
This occurs because the multiple transgenes cause double stranding of the RNA which leads to all the RNA being double stranded and therefore unable to be translated which leads to suppression of the gene product |
|
|
Term
| What is the biological role of co suppression? |
|
Definition
This is the phenomenon in plants n fungi that is when multiple transgenes are present, that particular gene is silenced
It occurs due to the fact that it helps prevent RNA viruses and against transposable element movement |
|
|
Term
| What is RNA interference? |
|
Definition
This is a method functional genomics that is for C. elegans and mammalian cells in culture
It uses dsRNA (200+ nts) which will correspond to the gene in question. The dsRNA is cleaved by DICER to create siRNA (Small interfering RNA, about 25nt with a 1nt sticky end on both 3' ends)
The siRNA will bind to a RNA induced silencing complex to create a targeting RISC. It targets the specific gene in question and degrades the RNA for the gene. This then results in silencing go the gene, genotype and phenotype.
Degrades due to a ribonuclease slicer which cuts the RNA in half
THIS IS CALLED POST TRANSCRIPTIONAL GENE SILENCING |
|
|
Term
|
Definition
It is an enzyme in the RNase III family which will cleave dsRNA in the RNA interference method of functional genomics.
It requires ATP |
|
|
Term
|
Definition
RNA-induced silencing complex
Used in RNA interference to take on the siRNA and degrade the gene in question to stop the gene product being translated
Contains an Ago protein which is the key component with two key domains, PAZ (binds to the siRNA) and PIWI (has the endonuclease activity to cut the target RNA) |
|
|
Term
| How do you administer RNAi into C. elegans? |
|
Definition
Inject it, feed it E. coli expressing the dsRNA needed on soak in solution containing the dsRNA
This feeding method was the main method used to determine the genome function of the C. elegans (85% of the genome) |
|
|
Term
| How can RNAi can be administered into mammalian cells? |
|
Definition
dsRNA cannot be as long as they are when put into C. elegans as the long strands will cause anti-viral response and is broken down
The singular siRNAs are placed in one by one or are expressed one by one as not to cause an issue |
|
|
Term
| What is the CRISPR-Cas9 system? |
|
Definition
It is an engineered nuclease that was derived from Streptococcus pyogenes (bacterial species). Originally for breaking down foreign viruses, it can be used in plants and animals to target specific breaks in DNA at specific sequences which allows for genome editing. Do this by adding modified template DNA strands in so that that strand is then accepted into the repairing DNA strand
The Cas9 is the protein/enzyme in question
Will only work if you add the template DNA, if not, the repair will just be fixed like any other break in the DNA. This is an issue of homology or non homology |
|
|
Term
| How is the nuclease of the CRISPR-Cas9 system made and how does it act in the Streptococcus pyogenes? |
|
Definition
There is a small stretch of RNA called tracrRNA which has a complementary sequence to a small stretch of direct repeat sequence from the Cas9 gene which is called the crDNA. This has a longer stretch of Spacer DNA attached which is the complementary sequence to where the nuclease is going to cut.
Looks like this:
[image]
This is manipulate in the CRISPR-Cas9 targeting sequence to look like this:
[image]
And the Spacer DNA is changed to be whatever piece of DNA that you are targeting and wanting to manipulate.
This whole thing is called gDNA |
|
|
Term
| What is gDNA in the CRISPR-Cas9 active nuclease targeting system? |
|
Definition
| This is the thing that when added to the Cas9 protein is the final active nuclease product containing just 1 long piece of manipulated RNA from the 'target gene spacer' RNA, the small piece of related DNA and tracrRNA |
|
|
Term
| How many nucleotides are there for the targeting of the gDNA in CRISPR-Cas9 targeting piece of RNA? |
|
Definition
| 20 nucleotides that are complementary to the site that you want to cut with the active nuclease |
|
|
Term
| How can the gDNA be manipulated further than just editing the targeting DNA sequence to elicit different reactions in the target DNA? |
|
Definition
You can change the domain activity of the Cas9 protein to either cut one or two DNA strands in the target DNA.
This will either create a clean cut or a nick (nickase activity) and this allows for different analysis that we do not need to know. |
|
|
