Term
Recognize groups of bases (motifs), from 4-8, in a specific manner.
Sequences arranged in palindromes.
Make double stranded breaks.
Many of these enzymes generate DNA fragments with “sticky ends”.
Also allow highly specific maps of the organismal genome to be made.
“Sticky ends” allows unrelated pieces of DNA to be put together. This is relevant to cloning foreign DNA. |
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Definition
| Restriction Endonucleases |
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Term
Plasmid – circular DNA molecule that can replicate in bacteria
Cosmid – vector based on bacteriophage gamma
BAC – bacterial artificial chromosome (F-factor plasmid)
PAC – P1 artificial chromosome (P1 bacteriophage)
YAC – Yeast artificial chromosome (Yeast chromosome replication start)
are all types of ??? |
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Definition
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Term
| Studying genes in a test tube is called ??? |
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Definition
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Term
| Living organisms are used in the experiments. |
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Definition
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Term
| Studied directly in the living organism, without removing. |
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Definition
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Term
| DNA segment capable of autonomous replication |
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Definition
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Term
| DNA that has been combined |
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Definition
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Term
| A system where a gene is placed back into cells and studied |
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Definition
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Term
In genetic engineering terminology, this is the act of putting DNA into a cell (bacterial, or other). This definition is different than the oncogenic term "transformed". Also, transfected, is a term with the same meaning although technically one transfects with a viral vector. The various transfection procedures are:
•CaP: incubation with a Calcium-phosphate solution.
Electroporation: Electric shock.
Lipofection: incubation with lipids. |
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Definition
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Term
| When exact multiple copies of a plasmid are available, it is said to be ???, and the copies are called ???. |
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Definition
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Term
| Totally undifferentiated stem cells that are not committed to any developmental pathway. ES cells. |
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Definition
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Term
| The total chromosomal content of a cell |
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Definition
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Term
| YAC = Yeast artificial chromosome |
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Definition
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Term
| Converts single stranded mRNA into double stranded DNA (cDNA -copy DNA). |
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Definition
| RT: Reverse Transcriptase |
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Term
| Reagent that is capable of recognizing a desired sequence in a complex mixture of many different sequences |
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Definition
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Term
| Short single stranded DNA molecule. |
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Definition
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Term
| A collection of DNA clones |
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Definition
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Term
| RFLP = Restriction fragment length polymorphisms. Variations in the length of specific restriction fragments. |
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Definition
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Term
| Technology for manipulating genes in the whole animal. |
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Definition
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Term
| An organism composed of cell of mixed origin. |
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Definition
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Term
| A whole organism (e.g., mouse) in which a specific gene has been disrupted so as to remove that gene function in the organism. |
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Definition
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Term
| Autonomously replicating bacterial minichromosome. Carry antibiotic resistance genes, which can be used to select among a bacterial population for those few bacteria that carry that specific ???. Thus, these bacterial genes are called marker genes. What bacterial vector has these characteristics? |
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Definition
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Term
| Plasmids bearing large foreign DNA segments are not stable in bacteria. This led to the establishment of various other vehicles for cloning large segments in bacteria. |
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Definition
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Term
| The entire central part of this phage was found not to be essential for replication in bacteria and could be deleted. The resulting shortened phage genome is however too short to be efficiently packaged into the phage coat. If foreign DNA is inserted into the middle of the two ends, the genome will now be efficiently packaged making a phage particle. Thus fragments of ~20 kilobases can be inserted, and a library of genomic DNA can be made. The entire human genome can be sampled for a given gene by screening one million phage. |
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Definition
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Term
It was found that only the single-stranded ends of lambda phage (Cos sites) are necessary for packaging. The cos sites were cloned into a plasmid forming ???. Pieces of DNA 35-45 kilobases can be cloned using this method. This vector can be used as a plasmid or as a viral vector, and packaged into a phage coat.
Sv40=eukaryotic virus
• This allows you to replicate virus in eukaryotic cells.
MCS = will insert foreign piece of DNA in this area. |
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Definition
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Term
Specific foreign DNAs can be isolated by looking for their expression in bacteria after cloning into plasmids developed to express (translate) the inserted foreign DNA
This allows you to screen for an antibody. |
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Definition
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Term
These Random Assortment of Foreign DNAs Cloned In Vectors Can Be:
o Copy/Genomic DNA
- One can synthesize a copy DNA (cDNA) molecule off of mRNA with reverse transcriptase
- This eliminates all unexpressed genes, focusing only on those genes that are expressed in that cell line (cDNA is entire complement of all Expressed genes)
o Then by attaching artificial restriction enzyme sites (linkers) to either end of the cDNA, it can than be inserted into a vector.
- The linkers allow the cDNA fragments to be RE digested and cloned. RE1 and RE2 are different RE sites, so one can directionally clone.
o If one does this for a population of mRNAs gathered from a particular cell type, the entire group of cloned cDNAs made from all the transcribed genes is called a cDNA library.
o Once cDNA probes for a specific gene are available, it is possible to look directly at the structures of the chromosomal gene themselves rather than at their mRNA transcripts. Only by examining much longer genomic fragments from chromosomes can regulatory sequences outside the transcribed region be seen. If one then clones the entire genome of a cell into a vector this set of clones is called a genomic library.
o As mentioned above, specific cDNAs can be isolated by looking for their expression in bacteria after cloning into vectors developed to express (translate) the inserted foreign DNA. An entire group of cDNAs from a particular cell type cloned into one of these vectors is called an expression library.
o Cloning the full complement of a cells genome, is called a Genomic Library |
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Definition
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Term
Once one has a random assortment of Cloned Foreign DNAs, to identify the particular piece of foreign DNA of interest, ONE NEEDS:
o To Identify & Obtain a PROBE:
A probe is a molecule made from either cloned DNA, or mRNA of the gene of interest,or an antibody that recognizes the gene product. If nucleic acid, then it is tagged either using radioactivity or flourescence for easy identification.
The crucial step in using recombinant DNA techniques is in obtaining this probe. Without a probe one cannot examine a gene or mRNA of interest because there is no way to identify that gene or message from the larger population of genes/messages in that cell.
Synthetic probes: Nucleic acid probes can also be made from the predicted sequence gleened from a sequence of amino acids, or made directly from mRNA sequence
When using an expression vector the resultant bacterial colonies are screened by probing with an antibody specific for the particular protein of interest.
With the cDNA library and the genomic library, one uses a nucleic acid probe. With an expression library, one uses an antibody probe. |
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Definition
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Term
o Hybridization vs. Expression Cloning
The next section will talk about hybridization, but I would like to include a comparison here
Hybridization: probe will hybridize to homologous piece of DNA. The colony will then label
Expression cloning: bacteria is sitting on the plate, hybridize to the bacterial colony. Instead of looking at all of them, can just take out the one that shows expression. |
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Definition
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Term
Using our Probe, the cloned Foreign DNAs can be screened by:
oHybridization or Blotting
- Given the existence of a probe, the structure of specific nucleic acids can be analyzed without prior cloning using hybridization technology.
- Hybridization is used to identify the cloned fragment of interest
• Single stranded DNA or RNA can be labeled with a fluorescent tag or radiolabeled.
• Must allow the temp to drop so that the 2 strands can reanneal
• 2 possible conditions:
o Stringent Hybridization: sequences have to be exact
o Reduced Stringency Hybridization: Not forming bonds everywhere |
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Definition
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Term
A Southern Hybridization or Blotting (DNA)
•High molecular weight DNA (genomic DNA) is cut with restriction enzymes. The resultant fragments are separated based on their size using gel electrophoresis.
o Gel Matrix
-When an electric field is applied to the gel matrix, the molecules will move towards the pole with the opposite charge (E.g., DNA is negatively charged so will run towards the positive charged pole).
-Additionally, the molecules will run faster the smaller they are, so the smallest(red) will go the furthest, etc.
•These separated fragments are then transferred onto filters and the filters probed with a tagged probe. When the filter is exposed to film only those fragments complementary to the probe are seen as bands on the film. This:
1) allows determination of sites for a given restriction enzyme.
2) allows determination of the number of copies of a gene in the genome. |
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Definition
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Term
Northern Hybridization (RNA)
• Total cellular RNA, or purified mRNA is separated and probed as in Souther Blotting. This technique is useful for research:
1) in comparing mRNA and cDNA sizes to determine if cDNA is full length.
2) in doing transcriptional analyses of a particular gene to examine things such as transcriptional regulation |
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Definition
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Term
Western Hybridization uses Proteins
•Proteins are separated by size using gel electrophoresis, transferred to a filter, and probed using antibodies |
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Definition
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Term
| True or False: Southern blotting is used in daignosis of a-thalassemias. Thalassemias are due to deletion of gene. Probe is hybridizes into region of DNA |
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Definition
True
REs fragment sites are deleted |
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Term
Use of Southern in diagnosis of sickle cell disease
- Sickle cell anemia is due to point mutation.
- AT is changed to
- In sickle cell this is not cleaved.
- Under homozygous normal, cleave DNA with mst2, get 3 cleavages
- In sickle cell, lost the cleavage site. Only have 2 mst2 in heterozygous. So get 2 fragements that hybridize,
- In diseased state, both chromosomes contain mutation so only get 1.35 kb fragment. Use this as way to diagnose pts.
- RFLP = restriction fragment length polymorphism. A change in restriction enzyme site. LINKED DIRECTLY to disease in this example
- Homozygous Normal - RNA extraction, cleavage by Mst 11, gell electrophoresis, transfer to nitrocellulose foil leads to 1.15 kb fragment
- Heterozygous (Sickle Cell trait) - same mechanism, leads to 1.15Kb and 1.35 kb fragments
- Heterozygous (Sickle Cell Disease) - produces 1.35 kb fragment |
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Definition
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Term
| Hemoglobin is composed of 2 alpha subunits and 2 beta subunits. Almost all of the mRNA from reticulocytes (immature red blood cells) codes for one of these two subunits. Thalassemias are family of related genetic diseases occurring in a population originating in the Mediterranean or Asia. They are due to mutations in either the alpha (a-thalassemia) or beta (b-thalassemia) subunit of globin. |
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Definition
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Term
RFLP and VNTR usage in DNA analysis
• Rflp = change in restriction site or due to deletion or insertion between restriction site
• Sequence repeats that occur over and over are allu repeats.
• Snps are even smaller and are just base changes upstream. They occur all the time so there are millions of them.
• Use allele specific probes.
• These probes can be used to sample from amniocentesis. |
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Definition
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Term
Prenatal Diagnosis of sickle cell disease by Southern Blotting with allele-specific oligonucleotide probes can lead to three patients.
1 - The fetus is homozygous for the normal gene
2 - The fetus is heterozygous for the normal gene and sickle cell mutation
3 - The fetus is homozygous for the sickle cell mutation |
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Definition
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Term
The use of dot-blotting for the diagnosis of a mutation with fluorescent-labeled probes for the normal and the mutated sequence. Denatured DNA from five different individuals is applied to two different nitrocellulose fibers, each in a single dot. One filter is dipped into a solution with a probe for the mutant sequence. Excewss probe is washed off and the bound probe is visualized under the UV lamp.
Easy Technique for quick diagnosis of mutation used in doctor's office |
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Definition
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Term
Northern Blotting Clinical Correlate
o Use in detecting mRNA changes leading to Menkes syndrome
Note: Presence of Normal mRNA does not necessarily mean you dont have Menkes syndrome |
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Definition
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Term
ONCE THE PIECE OF FOREIGN DNA OF INTEREST HAS BEEN IDENTIFIED, THE SEQUENCE OF THIS DNA CAN BE READ BY:
o NUCLEIC ACID SEQUENCING
- This procedure allows one to directly read the base sequence of a piece of nucleic acid. This can be done both for RNA and DNA. Currently, only the Sanger technique of sequencing is used. In this technique, one forms a reaction mix containing
- (1) a template DNA strand to be sequenced,
- (2) a short oligonucleotide primer sequence complementary to the 3' end of the template sequence,
- (3)a carefully controlled ratio of one particular 2',3'-dideoxynucleotide (e.g., ddATP) to the normal deoxynucleotide (dATP) plus the other three deoxynucleotides, and lastly add
- (4)DNA polymerase. Once DNA pol is added normal polymerization will begin from the primer moving 5' on the template strand. When this reaction gets to a T in our example, it will incorporate a dA at some frequency or a ddA at some frequency. If it incorporates a dA, the reaction will continue on that particular strand, but if it incorporates a ddA, the reaction will stop on that particular strand. If the correct ratio of ddATP to dATP is chosen, one will generate a series of labeled strands the lengths of which are dependent on the location of T relative to the end of the template strand. One creates 4 DNA reactions for each of the ddNTPs (A,T,G,C). These finished reactions are then separated by size on an acrylamide gel.
• There is no OH group on ddATP
Note --> The pattern of the generated fragments (complementary strand)is then read from the bottom of the gel to the top of the gel. |
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Definition
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Term
INTRODUCTION OF GENES INTO HIGHER EUCARYOTIC ORGANISMS
o Animals
- Introducing genes into tissue culture cells cannot directly reveal the molecular mechanisms responsible for the temporal and tissue-specific regulation of gene expression during early embryonic development.
- One way to approach this problem is to introduce genes or DNA into zygotes and subsequently analyze their pattern of expression, or their effect on the normal pattern of expression in embryonic tissues, or in the animals the fertilized egg gives rise to. Animals that develop from a fertilized egg with a foriegn gene insert, carry that gene in every cell,express it in some, and are referred to as transgenic animals.
1) The standard method of introduction of foreign DNA into the fertilized egg is by microinjection.
2) The foreign DNA is found in both somatic and germ-line cells indicating the integration event occurs very early in development before the cells have segregated into primordial germ cells and primordial somatic cells. This DNA can thus be transmitted through future generations of mice.
3) The extent of expression of foreign genes is also influenced by tissue differentiation and can be tissue-specific.
4) Appropriate foreign genes can be placed under the normal regulatory mechanisms of the host cells.
5) Knockout mice, gene disruption. (get homologous recombination) |
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Definition
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Term
(A) Biopharmaceuticals are the production of drugs using biotechnology in cells. Large amounts of human insulin are produced for use by all Type I and II diabetics in the world. It replaced extraction of insulin from bovine pancreases collected from slaughterhouses.
(B) Pharming is another way to produce large amounts of human proteins. However, here the human
protein is produced in the milk of large farm animals. Human Factor IX is used in treatment of hemophilia B and has been produced this way.
OF cells – ovine fetal cells.
This nuclear transfer procedure is what concerns
bioethicists and what we have seen discussed in the
news. As this techniques is perfected, the nucleus
could be from you, implanted in an enucleated
human oocyte, introduced into a pseudo-pregnant
woman producing a clone of you. |
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Definition
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Term
Antisense RNA
Another tool to study the structure and function of specific proteins. Antisense nucleic acids complementary to mRNAs will hybridize in situ (in the cell) and inactivate the mRNA through a number of mechanisms including blocking normal processing, and blocking ribosome binding. Antisense RNA can be introduced into a cell using common cloning techniques. One trick used is to substitute a phosphorothioate (phosphate and sulfur, PS) backbone in the oligonucleotides for a normal phosphate backbone. This PS backbone cannot be broken by cellular nucleases, and is very stable in cells.
Note; The gene fragment is clone backwards in relation to plasmid promoter, as compared to in original gene
CLINICAL CORRELATES:
- Opening new avenues in the control of disease processes. Phosphorothioate oligodeoxynucleotides now have reached phase I and II in clinical trials for the treatment of cancer and viral infections, so far demonstrating an acceptable safety and pharmacokinetic profile for continuing their development. The new drug Vitravene, based on a phosphorothioate oligonucleotide designed to inhibit the human cytomegalovirus (CMV), promises that some substantial successes can be reached with the antisense technique.
• Antisense RNA is working through siRNA. siRNa is targeting specific genes. Do not have to introduce dsRNA anymore, can just use siRNA. |
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Definition
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Term
Polymerase Chain Reaction (PCR)
• PCR allows the rapid production of large quantities of a specific DNA sequence.
• It requires two oligonucleotides that hybridize to the complementary DNA strands in a region of DNA that you want to amplify to clone the DNA, or for other purposes.
• These oligo's are added to a tube with the double stranded template DNA sequence of interest. The mixture is (1)heated to denature (melt) the entire DNA solution to single-stranded DNA. The primer has been added in vast excess. This means that when the solution is next (2)cooled to annealing temperature, each primer molecule will find its complementary sequence in the solution and hybridize to it.
• Then these hybridized oligonucleotides can serve as primers for a thermostable DNA polymerase (Taq)which can withstand very high temperatures that (3)extends each template strand. This series of events doubles the amount of template DNA in the tube.
• This DNA mix is then immediately subjected to the same series of events (1-3)in the same order, and through repeated cycling (each cycle of which doubles the amount of DNA template in the tube) one ends up with large amounts of a DNA molecule in hours as opposed to days or weeks when using cloning techniques.
• This can also be done with RNA sequences using a procedure called RT-PCR.
• In reference to the picture below, the FINAL PCR product will be the size of the primer.
CLINICAL CORRELATES:
This has many applications including diagnosis (e.g., AIDS), pedigree analysis (e.g., testing for genetic diseases like Huntingdons or hemophilia), forensic investigations, and evolutionary studies.
o Deletion scanning with PCR
- Exon x is missing. So when you amplify this up, you do not get a band.
o Use of PCR in diagnosis of a-thalassemias.
- Last time, we used probes for a-thalassemias
o Use of allele specific PCR in diagnosis of sickle cell.
- Before, used allele specific probes. Here using, allele specific PRIMERS
- Allele specific primer for sickle, will not recognize the normal
- Nonspecific primer recognizes both.
- So if primer is not there, get no product |
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Definition
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