Term
| Nucleic acid hybridizations |
|
Definition
| The process of establishing a non-covalent, sequence-specific interaction between two or more complementary strands of nucleic acids into a single hybrid |
|
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Term
|
Definition
| A fragment of DNA or RNA of variable length (usually 100-1000 bases long), which is used in DNA or RNA samples to detect the presence of nucleotide sequences (the DNA target) that are complementary to the sequence in the probe. The probe thereby hybridizes to single-stranded nucleic acid (DNA or RNA) whose base sequence allows probe-target base pairing due to complementarity between the probe and target. The labeled probe is first denatured (by heating or under alkaline conditions such as exposure to sodium hydroxide) into single stranded DNA (ssDNA) and then hybridized to the target ssDNA (Southern blotting) or RNA (northern blotting) immobilized on a membrane or in situ. |
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Term
| How do the environmental conditions determine the hybridization of nucleotide molecules? |
|
Definition
| Under very stringent hybridization conditions (e.g. very high temperatures, 42C), molecules can only hybridize to its perfect complement whereas under less stringent conditions (e.g. lower temperatures, 35C) a molecule is able to hybridze to molecules that are only related in sequence. |
|
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Term
| How can subtle differences in DNA sequence effect the hybridization between a probe and a template? |
|
Definition
| The effeciency with which two molecules hybridize is directly related to the match. Two molecules are less likely to hybridize under stringent conditions if their sequences are not perfect complements. |
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Term
|
Definition
Sequence specific DNA cutting enzymes which act as a method by which bacteria protect themselves from invading DNA. Restriction enzymes attack foreign DNA cutting it into small pieces. These bacteria protect their own DNA by
methylating bases in the recognition sequences for their restriction enzymes. |
|
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Term
| How do bacteria protect themselves from invading DNA? |
|
Definition
| Through the use of restriction enzymes, which attack foreign DNA by cutting it into small pieces. In order to protect their own DNA, bacteria methylate bases in the recognition sequences for their restriction enzymes. |
|
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Term
| What types of sequences do restriction enzymes frequently recognize? |
|
Definition
| Palindromic sequences: when the bases are read 5’ to 3’ the sequence is the same on both strands of the DNA (for example 5’-GGATCC-3’). |
|
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Term
|
Definition
| A map of known restriction sites within a sequence of DNA. Restriction mapping requires the use of restriction enzymes. |
|
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Term
| Restriction fragment linked polymorphisms |
|
Definition
| A technique that exploits variations in homologous DNA sequences. It refers to a difference between samples of homologous DNA molecules that come from differing locations of restriction enzyme sites, and to a related laboratory technique by which these segments can be illustrated. In RFLP analysis, the DNA sample is broken into pieces (digested) by restriction enzymes and the resulting restriction fragments are separated according to their lengths by gel electrophoresis. |
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Term
| When restriction enzymes cleave DNA, what remains at the cleavage site? |
|
Definition
Either:
1) Evenly cleaved, blunt ends, or
2) 5' or 3' overhangs, or "sticky ends", which are very useful in DNA cloning. |
|
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Term
|
Definition
| A lab method used to separate nucleic acids by size. The nucleic acid is loaded at one end of a matrix (the gel) that serves as a molecular sieve, voltage is applied across the gel and the nucleic acid being very negatively charged, due to the phosphodiester backbone, tries to move toward the anode. The speed with which the nucleic acid moves through the sieve (gel) is reflective of its size (short faster than long). This leads to separation of nucleic acids in a complex sample by size with small molecules at the front edge of the gel (usually shown as the bottom of the gel) and larger ones moving up from there to the top of the gel. |
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Term
| What could you add to detect DNA in an electrophoresis gel? |
|
Definition
1) Dyes that fluoresce under UV light when they bind DNA such as ethidium bromide, and
2) If the DNA has been labeled with a radioisotope then the gel can be laid onto X-ray film for some period of time after which the film is developed revealing the locations of the size separated DNA molecules.
More recently we have begun to use phospho-imagers to visualize radioactively labeled nucleic
acids: these use a phosphorescent screen much like the old glow in the dark watch faces. |
|
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Term
| What are two common gel materials used for size separation in electrophoresis? |
|
Definition
1) Agarose (extracted from seaweed) - forms a loose matrix ideal for separating large nucleic acids (500 bps to a few mega-base pairs).
2) Acrylamide - forms a tight matrix ideal for the separation of 25 up to
1000 nucleotides. |
|
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Term
| When would you use a gel made of agarose? |
|
Definition
| For separating large nucleic acids, 500 bps to a few mega base pairs. |
|
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Term
| When would you use a gel made of acrylamide? |
|
Definition
| For separating 25-1000 nucleotides. |
|
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Term
| What usually follows gel electrophoresis? |
|
Definition
| Usually transfer of the nucleic acids onto a special (nitrocellulose) paper. Usually a strong base (high pH)is included in the transfer step to simultaneously denature the DNA. This process creates a copy of the gel with the nucleic acids immobilized on the paper in the relative positions they occupied within the gel matrix and ready to hybridize to any complementary piece of nucleic acid. The paper can then be probed by hybridization for fragments with homology to a sequence of interest. (Southern blotting.) |
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|
Term
|
Definition
Following gel electrophoresis, a
second electrophoretic transfer out of the gel and onto special paper (usually nitrocellulose paper) that has extremely high affinity for nucleic acids frequently occurs. Usually a strong base (high pH)is included in the transfer step to simultaneously denature the DNA. This process creates a copy of the gel with the nucleic acids immobilized on the paper in the relative positions they occupied within the gel matrix and ready to hybridize to any complementary piece of nucleic acid. The paper can then be probed by hybridization for fragments with homology to a sequence of interest.
The probe is usually “labeled” by synthesizing radioactive complementary strands using nucleic acid synthesis enzymes such as DNA polymerase. Typically, α-32P-dATP is included in the DNA synthesis reaction in order to obtain 32P-labeled double-stranded DNA that is then denatured and allowed to hybridize to the nitrocellulose copy of the gel. The probe identifies the location of its complement on the nitrocellulose, nonspecific binding is washed off of the gel and specific binding is visualized by laying the nitrocellulose paper on X-ray film or a phospho-imager screen. |
|
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Term
| Probing the DNA-containning nitrocellulose paper |
|
Definition
| The probe is usually “labeled” by synthesizing radioactive complementary strands using nucleic acid synthesis enzymes such as DNA polymerase. Typically, α-32P-dATP is included in the DNA synthesis reaction in order to obtain 32P-labeled double-stranded DNA that is then denatured and allowed to hybridize to the nitrocellulose copy of the gel. The probe identifies the location of its complement on the nitrocellulose, nonspecific binding is washed off of the gel and specific binding is visualized by laying the nitrocellulose paper on X-ray film or a phospho-imager screen. |
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Term
|
Definition
Cloning in molecular biology parlance refers to the isolation and amplification of a specific DNA fragment/sequence. Typically one uses
enzymes that cut DNA in a sequence
specific manner (restriction
endonucleases), the fragment of
interest is identified and isolated
usually by gel electrophoresis, ligated
into a cloning vector using a
bacteriophage enzyme called T4 DNA
ligase and the resulting products are
transformed into E. coli where they are
identified and amplified. |
|
|
Term
| What is the role of restriction endonucleases in cloning? |
|
Definition
Restriction enzymes can cut the DNA strands to leave 4, 3, 2, 1 or 0
(blunt ends) base pair overhangs all of
which can be connected to other DNA
molecules by DNA ligase. The most useful restriction enzymes cut asymmetrically leaving either
5’ or 3’ overhangs known as “sticky ends” because they can hybridize to similarly cut DNA ends and be efficiently connected by DNA ligase. Note that blunt end ligations work poorly and sticky end ligations only work well if the overhangs are complementary. |
|
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Term
| What conditions must be met to ligate two strands of DNA cleaved by restriction enzymes? |
|
Definition
| You need "sticky ends" (5' or 3' overhangs) of complimentary base pairings and ATP to be paired by T4 DNA ligase. |
|
|
Term
| What joins two DNA molecules cleaved by restriction enzymes? |
|
Definition
|
|
Term
|
Definition
| DNA ends created by digestion with restriction enzymes can be connected to compatible DNA ends (for example in a vector) by using a T4 Bacteriophage (a bacterial virus) DNA ligase. This, and related enzymes, use the energy of ATP to seal the nicks in both strands of the annealed DNA connecting the 5’ phosphate of the deoxyribose moiety on one base to the 3’ hydroxyl of the deoxyribose moiety on the next base. This reaction is analogous to the ligation of DNA ends created during DNA replication. |
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Term
|
Definition
Molecular biologists commonly refer to the vehicles used to move, manipulate and amplify DNA sequences as vectors. All vectors are based upon naturally occurring DNA elements used by various organisms to transfer genetic information. The choice of vector is typically dictated by the organisms in which the investigator wants to introduce the DNA of interest and the size of the DNA fragment to be manipulated. The table below lists the commonly used cloning vectors and some of their characteristics. Examples are as follows:
1) Plasmids (up to 15 kb)
2) Bacteriophage lambda (up to 20 kb)
3) Cosmids (up to 45 kb)
4) BACs (100 to 300 kb)
5) VACs (100 to 2000 kb) |
|
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Term
| What are five examples of vectors? |
|
Definition
1) Plasmids (up to 15 kb)
2) Bacteriophage lambda (up to 20 kb)
3) Cosmids (up to 45 kb)
4) BACs (100 to 300 kb)
5) VACs (100 to 2000 kb) |
|
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Term
| How much exogenous DNA can be carried by plasmids? |
|
Definition
|
|
Term
| How much exogenous DNA can be carried by bacteriophage lambda? |
|
Definition
|
|
Term
| How much exogenous DNA can be carried by cosmids? |
|
Definition
|
|
Term
| How much exogenous DNA can be carried by BACs? |
|
Definition
|
|
Term
| How much exogenous DNA can be carried by VACs? |
|
Definition
|
|
Term
| Insertion of a DNA fragment into a plasmid (diagram) |
|
Definition
|
|
Term
| How can bacterial plasmids be used in cloning? |
|
Definition
Plasmids are are small closed circles
that carry an antibiotic resistance gene
(typically for ampicillin resistance or for tetracycline resistance) and a bacterial origin of replication. Following ligation of a DNA fragment of interest into a plasmid, the DNA is “transformed” into a laboratory
host bacterial strain. This procedure
bypasses the need for a bacterial pilus and simply involves treating the bacteria with chemicals that make them susceptible to picking up DNA from their environment. Once inside the bacterial cell, the plasmid is amplified by DNA replication, the antibiotic resistance gene is expressed and positive transformants are identified merely by plating the transformation reaction on medium that contains the appropriate antibiotic.
Since plasmid copy number can reach very high levels in the bacteria, large amounts of the plasmid can be prepared and analyzed, by restriction digests, for the presence of the exogenous
DNA fragment of interest. Plasmids are limited in the amount of human DNA they can carry (maximally 15 kilobases) and are therefore only useful for carrying fragments of human genes or cDNAs. |
|
|
Term
| How can bacteriophage lambda be used in cloning? |
|
Definition
Bacteriophage lambda is a bacterial virus. Its genome is a ~45 kb double stranded linear DNA molecule that becomes packaged in an infective particle. When introduced into a bacterium, the virus hijacks the cell to produce large amounts of its genome (by rolling circle replication) and coat proteins, the particles assemble incorporating one copy of the genome and the bacterium is induced to lyse releasing millions of the viral articles which can then go on to infect other cells. This life cycle produces tremendous amounts of viral particles that can be harvested and purified from the medium of infected bacterial cells. The virus carries genes for the amplification of its genome, the synthesis of its coat proteins and sequences required to package the DNA into the viral particles. Nonessential regions in the middle of the viral
genome can be removed and replaced by DNA cloning with exogenous DNA fragments of
interest. This allows the researcher to insert human DNA fragments up to ~20 kb in size. Following ligation of the exogenous DNA into lambda DNA, the product is transformed into the bacteria and a viral infection ensues in the cells that pick up the modified lambda DNA. |
|
|
Term
| How can cosmids be used in cloning? |
|
Definition
| Cosmids are essentially gutted versions of Bacteriophage lambda; most of the essential genes have been removed from the genome leaving only the sequences at the end required to replicate the DNA and package the DNA into viral particles. Since the viral particle can only package ~45 kb of DNA, this leaves more room to put in human DNA by cloning (nearly 45 kb). One merely ligates the short end sequences (right and left arms) of the bacteriophage lambda genome onto the ends of the DNA fragment of interest and transforms the ligation product into a “helper” strain of bacteria. The helper strain carries and expresses the essential genes of the Bacteriophage lambda allowing for a productive infection in which the cosmid DNA (with cloned insert) is amplified and packaged into viral particles that can then be isolated from the medium of lysed cells. |
|
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Term
| How can bacterial articifial chromosomes (BACs) be used in cloning? |
|
Definition
| Bacteria can be fooled into carrying large pieces of human DNA (100-300 kb) in a plasmid (double stranded circle) form as artificial chromosomes or BACs. This is probably the most useful and stable way to maintain large pieces of human genomic DNA. |
|
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Term
| How can yeast articifial chromosomes (YACs) be used in cloning? |
|
Definition
The sequence elements required to stably
maintain yeast (S. cerevisiae a.k.a. bread and beer yeast) chromosomes are well understood/described and include telomeres at the ends, a centromere for mitotic segregation and origins of
replication. These sequence elements can be attached to very large pieces (100-2,000 kb) of human genomic DNA
(by DNA cloning) and when introduced by transformation into yeast cells, the cloned DNA will be maintained as if it were a normal yeast chromosome, hence the moniker Yeast Artificial Chromosomes or YACs. The one disadvantage of YACs is that yeast have extremely high levels of homologous
recombination which can lead to scrambling of human DNA via recombination between the many repetitive DNA elements found in human DNA (ALUs and LINEs). |
|
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Term
| What is the most useful and stable way to maintain large pieces of human genomic DNA? Why? |
|
Definition
| BACs. Yeast have extremely high levels of homologous recombination which can lead to scrambling of human DNA via recombination between the many repetitive DNA elements found in human DNA (ALUs and LINEs). |
|
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Term
| What is the one disadvantage of YACs? |
|
Definition
| Yeast have extremely high levels of homologous recombination which can lead to scrambling of human DNA via recombination between the many repetitive DNA elements found in human DNA (ALUs and LINEs). |
|
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Term
| Where does recombination often take place in yeast? |
|
Definition
| Between the many repetitive DNA elements found in human DNA (ALUs and LINEs). |
|
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Term
|
Definition
To construct a genomic library, one isolates the genomic DNA of interest
and fragments that DNA by digesting
with restriction endonucleases.
Typically the size of the fragments needs to be controlled and this is achieved by the choice of enzymes employed (infrequent cutters give larger fragments, frequent cutters smaller fragments). If one desires very large fragments, the restriction enzyme is not allowed to cut to completion (a partial digest), the fragments are then size selected and purified by gel electrophoresis. The random fragments can then be ligated into a cloning vector (plasmid, phage, cosmid, YAC or BAC), transformed into a host (bacteria or yeast) and several million or more individual clones are collected and preserved in the freezer. |
|
|
Term
| Construction of a genomic DNA library (diagram) |
|
Definition
|
|
Term
| Construction of a cDNA library (diagram) |
|
Definition
|
|
Term
|
Definition
Since much of the human genome is not expressed and of the amount that is expressed much of the sequence is removed by splicing, genomic libraries can be of limited usefulness. For example, a single human gene can cover many 100s of kilobases of genomic sequence of which only 1% or less may become the final mRNA. It may be difficult to get a single clone carrying
the gene and very difficult to identify all of the exons in that DNA. To circumvent these problems one can construct a library from the mRNA molecules expressed in any given tissue. In the first step, RNA is isolated from the tissue of interest and
the mRNA is enriched by purifying the RNA with poly(A) tails (using an oligo dT affinity column). An oligo dT primer is then annealed to the poly(A) tail and an enzyme called reverse
transcriptase (first identified in retroviruses) uses the dT primer to synthesize a complementary DNA strand. The RNA is then degraded and the single-stranded DNA copy is used as a template by a DNA polymerase to synthesize the second strand. The double stranded DNA copies of the mRNAs (called cDNAs) are then ready to be ligated into a cloning vector (usually a plasmid vector) and transformed en masse into bacteria, several thousand individual clones are collected and preserved. |
|
|
Term
| When considerations must be taken when using cDNA libraries? |
|
Definition
| One must remember that the abundance of a clone in a cDNA library reflects that gene’s expression level in the tissue from which the original mRNA was purified. Therefore, if the gene you are looking for is expressed at very low levels or not at all in the tissue used to isolate the RNA, it will be under represented and very difficult to isolate from that library. |
|
|
Term
| What are the steps involved in constructing a cDNA library? |
|
Definition
1) RNA is isolated from the tissue of interest and the mRNA is enriched by purifying the RNA with poly(A) tails (using an oligo dT affinity column).
2) An oligo dT primer is then annealed to the poly(A) tail and an enzyme called reverse transcriptase (first identified in retroviruses) uses the dT primer to synthesize a complementary
DNA strand.
3) The RNA is then degraded and the single-stranded DNA copy is used as a template by a DNA polymerase to synthesize the second strand.
4) The double stranded DNA copies of the
mRNAs (called cDNAs) are then ready to be ligated into a cloning vector (usually a plasmid vector) and transformed en masse into bacteria, several thousand individual clones are collected and preserved. |
|
|
Term
| Screening a library with a radioactive probe (diagram) |
|
Definition
|
|
Term
|
Definition
| Repeated rounds of primer directed DNA replication off of a rare template. Each round of the reaction produces more copies of the template that can be primed in the next reaction leading to an exponential amplification of the template molecule. The boundaries/size of the template are defined by the choice of the primers. The key to the procedure is the use of DNA Polymerases from thermophilic (heat loving) micro-organisms that can survive repeated treatments at 95°C (just below boiling). |
|
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Term
|
Definition
| DNA Polymerases from thermophilic (heat loving) micro-organisms that can survive repeated treatments at 95°C (just below boiling). |
|
|
Term
| What are the steps in a PCR reaction? |
|
Definition
1) All of the DNA molecules are denatured (separated) by heating to 95°C.
2) The reaction temperature is lowered
to allow the primers to anneal to their template single stranded DNA targets and the polymerase is allowed to extend 5’-3’.
3) This simple process is then repeated in subsequent cycles, each time doubling the number of template DNA molecules. |
|
|
Term
| What is required to begin a PCR reaction? |
|
Definition
1) One or more of the dsDNA molecules that contains a few copies of the sequence the investigator wishes to amplify.
2) 2 primers, one complmentary to a region to the right on the top strand of DNA and the other complementary on the left region of the bottom strand.
3) A thermophilic DNA polymerase.
4) Buffer.
5) The 4 precursor deoxynucleotides. |
|
|
Term
| How does one analyze PCR results? |
|
Definition
| By analyzing the PCR reaction by agarose gel electrophoresis. Size standards are run next to the sample to estimate size and different (known) quantities of similarly sized DNA can be run on the gel to estimate quantity of product. Since each cycle does not work perfectly, it is very tricky business to back extrapolate (using the above equation) to get the number of starting DNA template molecules. However, if one uses the appropriate controls (for example running a set of standard curve reactions) you can estimate the relative amounts of starting template between two or more samples. |
|
|
Term
| What is the best way to quantify non-abundant DNA sequences? |
|
Definition
| Quantifying non-abundant DNA sequences in samples is best done by a “Real Time” PCR instrument: this machine uses fluorescence to quantify product production at each step of the reaction(Real Time). The resulting curve of product versus time (cycles) can be used to accurately compare template abundance in different samples. |
|
|
Term
|
Definition
| A single sample can be simultaneously analyzed with multiple sets of primers (Multi-Plex PCR). One merely adds the primer sets to the same PCR reaction. This type of experiment needs to be carefully designed so that all of the products can be separated from each other by gel electrophoresis. In fact, clinicians have designed many diagnostic tests to simultaneously analyze a single sample for the presence of multiple alleles. Obviously, this saves a tremendous amount of time and money. |
|
|
Term
| What is the difference between PCR and multiplex PCR? |
|
Definition
| PCR targets only a single gene at a time, whereas the addition of multiple sets of primers in multiplex PCR allows for the targeting of multiple genes at once. |
|
|
Term
Exon analysis of the Dystrophin gene by
multi-plex PCR (diagram) |
|
Definition
| Muscular Dystrophy is frequently caused by deletions of one or more of the exons for the Dystrophin gene, whose product stabilizes the sarcolemma of muscle cells. To analyze for exon deletions, multi-plex PCR is done with primers designed to amplify subsets of the dystrophin gene exons. On the bottom of the figure is shown the structure of the dystrophin gene and the locations of the primers used. The top of the figure shows an agarose gel of multi-plex PCR reactions on the DNA from a normal individual and from three patients with muscular dystrophy. As you can see, Patient 1 is lacking exons 45-48, Patient 2 is lacking exon 48, and Patient 3 is lacking exon 44. Note that a complete description of each patient’s breakpoints would require additional analysis. |
|
|
Term
| PCR to detect the presence of HIV or other pathogens (diagram) |
|
Definition
| This variation of the procedure is called RTPCR for reverse transcriptase PCR. |
|
|
Term
| How can you use PCR to detect the presence of HIV or other pathogens? |
|
Definition
| In the RT-PCR test, viral RNA is extracted from the patient's plasma and is treated with reverse transcriptase (RT) to convert the viral RNA into cDNA. The polymerase chain reaction (PCR) process is then applied, using two primers unique to the virus's genome. After PCR amplification is complete, the resulting DNA products are hybridized to specific oligonucleotides bound to the vessel wall, and are then made visible with a probe bound to an enzyme. The amount of virus in the sample can be quantified with sufficient accuracy to detect threefold changes. |
|
|
Term
| How can PCR allow cloning of a piece of DNA (diagram)? |
|
Definition
| Fragments of genomic DNA can be amplified by PCR (panel A at right) or one can amplify cDNA corresponding to part, or all of the coding region of a gene (panel B at right). In the latter case one merely isolates mRNA and uses Reverse Transcriptase to make a DNA copy of the mRNA corresponding to the gene of interest prior to amplification by a standard PCR reaction (RT-PCR). Typically, Restriction Enzyme recognition sites are placed at the beginning of the primers to facilitate ligation of the product into the cloning vector of choice. |
|
|
Term
| Where along the process can gene expression be analyzed? |
|
Definition
1) Transcription to produce mRNA.
2) Translation to produce protein. |
|
|
Term
| How do you purify mRNA for a Northern blot? |
|
Definition
| Since mRNA has a poly(A) tail, this is simply done by purifying on a oligo d(T) affinity resin. The RNA is incubated with an insoluble resin to which oligo d(T) has been covalently coupled, the mRNA hybridizes to the oligo d(T) via its poly (A) tail, the resin is washed and the purified mRNA is eluted with high salt. |
|
|
Term
| What are Northern blots able to detect? |
|
Definition
|
|
Term
| How would you determine if the mRNA for a gene is present in a cell/tissue/patient? |
|
Definition
|
|
Term
| Using Northern blot to diagnose varying degrees of muscular dystrophy. |
|
Definition
| Northern blots can be used to determine if the mRNA for a gene is present in the cells/tissues/patient, the expression level of the mRNA, and if the mRNA is the proper size. Patients with severe muscular dystrophy may lack the mRNA for dystrophin in their muscle cells, while patients with less severe forms may express a smaller than normal mRNA for dystrophin due to exon deletion. |
|
|
Term
| If a protein has a lot of acidic amino acids (e.g. aspartic and glutamic acid), will it be positively or negatively charged at pH 7? |
|
Definition
|
|
Term
| If a protein has a lot of basic amino acids (e.g. lysine and arginine), will it be positively or negatively charged at pH 7? |
|
Definition
|
|
Term
| If you wanted to use gel electrophoresis to separate proteins based on size alone, what would you do? |
|
Definition
| You have to make the proteins' charge irrelevant. This is done by heating the protein sample in the presence of the detergent, sodium dodecyl sulphate (SDS). The SDS is negatively charged and quantitatively coats the peptide backbone of the proteins, denaturing the proteins and giving them an overwhelmingly negative charge. Typically a strong reducing agent such as ß-mercaptoethanol is included to also reduce any disulfide bonds. This form of protein electrophoresis is called denaturing protein electrophoresis. |
|
|
Term
| Denaturing protein electrophoresis |
|
Definition
| A type of protein electrophoresis where the protein's charge is rendered irrelevant and the molecules are separated based on size alone. This is done by heating the protein sample in the presence of the detergent, sodium dodecyl sulphate (SDS). The SDS is negatively charged and quantitatively coats the peptide backbone of the proteins, denaturing the proteins and giving them an overwhelmingly negative charge. Typically a strong reducing agent such as ß-mercaptoethanol is included to also reduce any disulfide bonds. |
|
|
Term
| What is added to a protein sample to render the protein's charge irrelevant in denaturing protein electrophoresis? |
|
Definition
| Sodium dodecyl sulphate (SDS) while denaturing the protein. |
|
|
Term
| Sodium dodecyl sulfate (SDS) |
|
Definition
| Detergent added to protein sample while denaturing which renders the protein's charge irrelevant so that it can be sorted by size only in denaturing protein electrophoresis. |
|
|
Term
| What would you use to reduce any disulfide bonds when running denaturing protein electrophoresis? |
|
Definition
|
|
Term
|
Definition
| A strong reducing agent that reduces disulfide bonds in denaturing protein electrophoresis. |
|
|
Term
| Which assay would you perform to compare charges between two proteins? |
|
Definition
| Non-denaturing protein electrophoresis. It's the same as denaturing protein electrophoresis minus the heating, SDS, and mercapto-ethanol. Protein separation then is based solely on the charge and shape of the protein. |
|
|
Term
| Non-denaturing protein electrophoresis |
|
Definition
| An assay performed to compare the charges between two proteins. It is performed the same way as denaturing protein electrophoresis minus the heating, SDS, and mercapto-ethanol. Protein separation then is based solely on the charge and shape of the protein. |
|
|
Term
| What's the difference between denaturing protein electrophoresis and non-denaturing protein electrophoresis, and when would you perform each? |
|
Definition
| Denaturing protein electrophoresis employs the use of SDS, ß-mercaptoethanol, and heat to render the protein's charge irrelevant and instead sort proteins by size only. You would then use non-denaturing protein electrophoresis if you wanted to sort proteins based on charge and shape of the protein. |
|
|
Term
| Isoelectric focusing (IEF) |
|
Definition
| A method of gel electrophoresis used to separate proteins based on charge. In this technique, a stable pH gradient is established in the gel matrix, the protein is loaded at one end of the gel and a voltage is applied. The proteins will migrate in the gel until they reach that point at which they have no net charge; this is called the protein’s isolelectric point or pI. |
|
|
Term
|
Definition
| The point in the gel at which they have no net charge. |
|
|
Term
| Two-dimensional gel electrophoresis |
|
Definition
| Involves both IEF and denaturing gel electrophoresis. First, the isolelectric focusing is done in a tube gel then the tube gel is placed on top of a standard denaturing gel. This method has such good resolving power that it can be used to describe the entire proteome of a cell or tissue. |
|
|
Term
| Isoelectric focusing (diagram) |
|
Definition
|
|
Term
|
Definition
| Detection and analysis of proteins is typically done using antibodies specifically reactive to the protein under study. The antibodies are usually “raised” in animals such as rabbits, mice, goats etc by injecting the animal with large amounts of the protein, or fragments of the protein, thereby simulating an infection. The animal mounts an immune response to the protein antigen, their B-cells make and secrete large amounts of antibody specific to the protein which can then be purified from the serum of the animal. The antibody specific to the protein of question is typically called the primary antibody. |
|
|
Term
|
Definition
| Reacts with and binds to primary antibody. A tag is often covalently coupled to the secondary antibody so its location and abundance can be measured. |
|
|
Term
| How are secondary antibodies made? |
|
Definition
Most assays also require a secondary antibody that will react to and bind the primary antibody. Animals will recognize the antibodies from other species as foreign and will amount an antibody response if challenged with antibodies from another species. This is how the secondary antibodies are made. For example, if your primary antibody was made in a rabbit, an appropriate secondary antibody could be raised in a goat. Since the antibodies from one species will share a constant region, you only need raise the secondary antibody against the constant region found in the antibodies from the organism in which the primary antibody was raised. This secondary antibody will then react to all primary antibodies made in that species (rabbit in our example).
 |
|
|
Term
| What do Western blots measure? |
|
Definition
|
|
Term
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Definition
| Western Assays are performed much like Southern Blots. Protein is isolated from a patient by biopsy or from serum. The protein is then separated based on size by gel electrophoresis following which the proteins are transferred out of the gel onto nitrocellulose paper creating a paper copy of the gel. The paper is then incubated with the primary antibody that then binds the protein under study. The blot is washed and incubated with a secondary antibody that has been coupled to an enzyme (usually Horseradish Peroxidase or HRP). Finally the blot is incubated in a substrate for HRP that gives off light when acted upon by a peroxidase and the protein location and abundance is visualized by exposing the blot to film. |
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Term
| Becker muscular dystrophy |
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Definition
| A fairly milder form of Duchenne muscular dystrophy characterized by reduced amounts of the protein dystrophin and dystrophin not of the correct size (due to exon deletion). |
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Term
| Becker muscular dystrophy |
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Definition
| A fairly milder form of Duchenne muscular dystrophy characterized by reduced amounts of the protein dystrophin and dystrophin not of the correct size (due to exon deletion). |
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Term
| What can immuno-flourescence microscopy tell you? |
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Definition
| Western Assays can tell you if a protein is expressed and if it is of the proper size. Additional information such as proper cellular expression and localization can be obtained by immuno- fluorescence microscopy. Cells or a tissue section are incubated with the primary antibody followed by a secondary antibody to which a fluorescent tag has been coupled. The sample is then placed on a microscope, illuminated with the excitation wavelength for the fluorescent tag and the location of the protein is visualized by looking at the emission wavelength for the fluorescent tag. In this manner, the investigator can determine if the protein is expressed, what cells it is expressed in, and if the protein is properly localized within those cells. |
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Term
| Immuno-fluorescence microscopy of muscle biopsies from patients with Muscular Dystrophy (figure) |
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Definition
| The example above shows such an analysis for Dystrophin in muscle biopsies from a normal individual versus two patients with Muscular Dystrophy. Note the reduced expression in the patient with Beckers and the complete absence in the patient with Duchennes Muscular Dystrophy. |
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Term
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Definition
| The process by which double stranded RNA molecules corresponding to a cellular gene could be used to down regulate the expression of that gene as a mechanism by which cells protect themselves against RNA viruses and rampant retro-transposon amplification. |
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Term
| Discuss the steps involved in RNA interference |
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Definition
1) The response is initiated by introducing double-stranded, complementary RNA into the cell, organism or tissue (you must introduce genes coding for both strands of the RNA - this is frequently done by constructing a gene that will express a self-complementary RNA that will form a large “hairpin”).
2) The hairpin gene is then introduced into the cells or tissues with viral or plasmid based vectors.
3) Once the dsRNA enters the cell it is recognized by a protein complex that contains an RNA endonuclease called “Dicer” in conjunction with an RNA helicase. This complex cuts the dsRNA into small (~23 nucleotide) pieces that then direct, via homology, the complex to a complementary cellular mRNA species.
4) The RNA endonuclease Dicer then cuts the mRNA molecule leaving it susceptible to further destruction by abundant cellular exonucleases.
The RNA-protein complex can act catalytically to destroy many mRNA molecules and the short RNA products can be replicated to amplify the RNA interference response. In this manner, RNAi can be propagated to progeny cells and transferred to neighboring cells resulting in a possibly prolonged and broad response. |
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Term
| What type of molecule initiates the RNAi response? |
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Definition
| The introduction of dsRNA into a cell, tissue, or organism. |
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Term
| How is the hairpin gene introduced into cells or tissues? |
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Definition
| The hairpin gene is introduced into the cells or tissues with viral or plasmid based vectors. Once the dsRNA enters the cell it is recognized by a protein complex that contains an RNA endonuclease called “Dicer” in conjunction with an RNA helicase. This complex cuts the dsRNA into small (~23 nucleotide) pieces that then direct, via homology, the complex to a complementary cellular mRNA species. The RNA endonuclease Dicer then cuts the mRNA molecule leaving it susceptible to further destruction by abundant cellular exonucleases. The RNA-protein complex can act catalytically to destroy many mRNA molecules and the short RNA products can be replicated to amplify the RNA interference response. |
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Term
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Definition
| An RNA endonuclease which, along with a complex including protein and an RNA helicase, cuts the dsRNA into small (~23 nucleotide) pieces that then direct, via homology, the complex to a complementary cellular mRNA species. The RNA endonuclease Dicer then cuts the mRNA molecule leaving it susceptible to further destruction by abundant cellular exonucleases. |
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Term
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Definition
| Green fluorescent protein - a protein with the property of absorbing light in the UV range and emitting light in the visible spectrum. The genes for these fluorescent proteins can be fused to any gene of interest such that the resulting protein product is a fusion between GFP and the protein of interest. This fusion protein can be expressed in individual cells or in whole animals so that protein expression and localization can be followed in living cells and in real time. |
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Term
| Deep/ultra deep DNA sequencing |
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Definition
| More recent developments have led to tremendous improvement in DNA sequencing capabilities such that it is now reasonable to sequence an individual’s genome in 1-2 days at a current cost of ~$5,000. A number of platforms exist that are collectively referred to as deep or ultra-deep sequencing instruments that are capable of generating up to 20 giga base pairs of sequence in a single run on the instrument. These instruments are opening the door to personalized medical genetics and are revolutionizing rare pathogen detection in human samples. The diagram below shows how the Illumina instrument functions. All of these instruments in general operate by affixing, amplifying, and sequencing single short DNA molecules in situ on a flow cell surface and much of the process is automated including assembly of whole genome sequence from millions of short sequence reads. |
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Term
| Describe the process of DNA microarrays |
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Definition
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