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VARIENTS OF PCR
ISOLATION OF A GENE BY PCR If the Primers anneals anneal both the sides of the gene of interest No selection required THEN WHY CLONING Sequence information is required Length of DNA sequence IF SEQ INFORMATION IS NOT KNOWN If we have information on heterologous gene / equivalent gene
Its important to isolate gene whose seq is known Fish out gene - based on flanking sequence information DNA polymorphism Allelic forms of genes can be isolated Diagnostics both in plant and animal including human - Early diagnosis
Forensic
We covered
Hot start PCR Nested PCR Touch down PCR Overlap extension
Multiplexing
.. and
LATE-PCR
A recent modification on this process, known as Linear-After-TheExponential-PCR (LATE-PCR), uses a limiting primer with a higher melting temperature (Tm) than the excess primer to maintain reaction efficiency as the limiting primer concentration decreases mid-reaction
Smiths Detection has an exclusive license for LATE-PCR from Brandeis University for all markets, worldwide This enables one to detect low numbers of target organisms
The oligonucleotides alternate between sense and antisense directions, and the overlapping segments determine the order of the PCR fragments, thereby selectively producing the final long DNA product
Inverse PCR
Commonly used to identify the flanking sequences around genomic inserts. It involves a series of DNA digestions and self ligation, resulting in known sequences at either end of the unknown sequence.
Ligation-mediated PCR: uses small DNA linkers ligated to the DNA of interest and multiple primers annealing to the DNA linkers; it has been used for DNA sequencing, genome walking, and DNA foot printing
LM-PCR is a method of sequencing directly from genomic DNA if you have a primer for the region of interest.
The DNA is chemically cleaved as if for Maxam Gilbert chemistry, producing a whole bunch of fragments that end in G, A, T or C.
Denature the DNA and let the primer anneal to the fragments that are related the region you are interested in.
By primer extension off that primer, you make only those fragments double stranded and blunt ended; these molecules are able to be ligated to a linker (the linker is staggered at one end, and blunt on the other, so the ligation is directional Now you have a bunch of fragments that have two known ends (your primer, and the linker) and you can use PCR to amplify the various fragments, run them on a gel and determine the sequence. The reason you might want to go to all this trouble would be to examine conditions of DNA that are lost upon cloning (e.g. methylation), or to examine footprints of DNA binding proteins in vivo.
BRIDGE PCR
Specific primer (SP) ested sequence specific primer complementary to vector sequence N High melting temperature, Tm=58-63 deg. C Arbitrary degenerate (AD) primer Relatively shorter
Universal Fast Walking: for genome walking and genetic fingerprinting using a more specific 'two-sided' PCR than conventional 'one-sided' approaches (using only one genespecific primer and one general primer which can lead to artefactual 'noise)
In silico PCR:
(Digital PCR, Virtual PCR, Electronic PCR, e-PCR) refers to computational tools used to calculate theoretical polymerase chain reaction results using a given set of primers(probes) to amplify DNA sequences from a sequenced genome or transcriptome
DD-PCR
To detect the differential expression of the gene Usually subtractive-hybridization and differential cDNA cloning
require large amounts of mRNA derived from cell or tissue samples, and are very laborious and time consuming, often taking many months from start to finish.
Alternatively
To verify the differential expression status of each of these sub-clones, Northern-Blot assays are performed using RNA isolated from the original sample sources
COLONY PCR
Colony PCR can be used to identify colonies where your favorite gene (yfg) has been replaced with a marker gene by homologous recombination, and to distinguish homologous recombination events from nonhomologous. It can also be used to identify colonies from a tetrad that carry a particular gene replacement
DNA ligase
Repairs gaps in the sugar-phosphate backbone of DNA
Creates phosphodiester bonds
Each ligated product as well as original target serve as a template A modification of this technique gapped LCR (G-LCR)
Kits are available for the detection of C.trachomatis, N. gonorrhoeae etc.
LCR
The ligase chain reaction (LCR) is a method of DNA amplification. While the better-known PCR carries out the amplification by polymerizing nucleotides, LCR instead amplifies the nucleic acid used as the probe. For each of the two DNA strands, two partial probes are ligated to form the actual one; thus, LCR uses two enzymes: a DNA polymerase and a DNA ligase. Each cycle results in a doubling of the target nucleic acid molecule. A key advantage of LCR is greater specificity as compared to PCR
Helicase-dependent amplification
Similar to traditional PCR, but uses a constant temperature rather than cycling through denaturation and annealing/extension cycles DNA helicase, an enzyme that unwinds DNA, is used in place of thermal denaturation
TMA/ NASBA
Detection of HIV, HCV, vericella zoster virus, cytomegalovirus, rhinivirus, measles, papillomavirus, M. tuberculosis, M. pneumoniae etc.
3-CAPuPyTG-5
SDA
REFERENCE
Dieffenbach, C.W and Dvksler, G.S. (1995) PCR primer: a laboratory manual. CSHL press, Cold Spring Harbor, USA. Erlich, H. A. 1989. PCR technology: Principles and applications for DNA amplification. Stockton Press, New York.
Innis, M. A., D. H. Gelfand, J. J. Sninsky, and T. J. White. 1990. PCR Protocols: A guide to methods and applications. Academic Press, New York.
Ellingboe, J., and U. B. Gyllensten. 1992. The PCR technique: DNA sequencing. Eaton Publishing Co., Natick, Mass. Bej, A. K., M. H. Mahbubani, and R. M. Atlas. 1991. Amplification of nucleic acids by polymerase chain reaction (PCR) and other methods and their applications. Critical Reviews in Biochemistry and Molecular Biology 26:301-334.
Journal: "PCR Methods and Applications" published quarterly by Cold Spring Harbor Press