Other Polymerase Chain Reac3on (PCR) Methods

Transcription

1 Other Polymerase Chain Reac3on (PCR) Methods

2 What is PCR? The Reaction Components 1) Target DNA - contains the sequence to be amplified. 2) Pair of Primers - oligonucleotides that define the sequence to be amplified. 3) dntps - deoxynucleotidetriphosphates: DNA building blocks 4) Thermostable DNA Polymerase - enzyme that catalyzes the reaction 5) Mg ++ ions - cofactor of the enzyme 6) Buffer solution maintains ph and ionic strength of the reaction solution suitable for the activity of the enzyme

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4 Varia3ons of the PCR Colony PCR Nested PCR Mul3plex PCR AFLP PCR Hot Start PCR In Situ PCR Inverse PCR Asymmetric PCR Long PCR Long Accurate PCR Reverse Transcriptase PCR Allele specific PCR Real 3me PCR

5 Colony PCR Colony PCR- the screening of bacterial (E.Coli) or yeast clones for correct liga6on or plasmid products. Pick a bacterial colony with an autoclaved toothpick, swirl it into 25 μl of TE autoclaved dh2o in an microfuge tube. Heat the mix in a boiling water bath (90-100C) for 2 minutes Spin sample for 2 minutes high speed in centrifuge. Transfer 20 μl of the supernatant into a new microfuge tube Take 1-2 μl of the supernatant as template in a 25 μl PCR standard PCR reac6on.

6 Hot Start PCR This is a technique that reduces non- specific amplifica6on during the ini6al set up stages of the PCR The technique may be performed manually by hea6ng the reac6on components to the mel6ng temperature (e.g., 95 C) before adding the polymerase Specialized enzyme systems have been developed that inhibit the polymerase's ac6vity at ambient temperature, either by the binding of an an3body or by the presence of covalently bound inhibitors that only dissociate aqer a high- temperature ac6va6on step DNA Polymerase- Eubacterial type I DNA polymerase, Pfu These thermophilic DNA polymerases show a very small polymerase ac3vity at room temperature.

7 Asymmetric PCR Asymmetric PCR is used to preferen6ally amplify one strand of the original DNA more than the other. It finds use in some types of sequencing and hybridiza3on probing where having only one of the two complementary stands is ideal. PCR is carried out as usual, but with a great excess of one primers for the chosen strand.

8 Nested PCR Two pairs (instead of one pair) of PCR primers are used to amplify a fragment. First pair - amplify a fragment similar to a standard PCR. Second pair of primers- nested primers (as they lie / are nested within the first fragment) bind inside the first PCR product fragment to allow amplifica6on of a second PCR product which is shorter than the first one. Advantage- Very low probability of nonspecific amplifica6on

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10 Mul3plex PCR Mul6plex PCR is a variant of PCR which enabling simultaneous amplifica6on of many targets of interest in one reac6on by using more than one pair of primers.

11 Reverse Transcriptase PCR Based on the process of reverse transcrip6on, which reverse transcribes RNA into DNA First step of RT- PCR - "first strand reac6on - Synthesis of cdna using oligo dt primers (37 C) 1 hr Second strand reac6on - Diges6on of cdna:rna hybrid (RNaseH)- Standard PCR with DNA oligo primers Allows the detec6on of even rare or low copy mrna sequences by amplifying its complementary DNA

12 Real Time PCR Real Time PCR is a technique in which fluoroprobes bind to specific target regions of amplicons to produce fluorescence during PCR. The fluorescence, measured in Real Time, is detected in a PCR cycler with an inbuilt filter flurometer.

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14 Standard Curve for Absolute quantification Good efficiency, good sensitivity and good predictive power.