Lectut btn-202-ppt-l28. variants of pcr-ii

Biotechnology Engineering

1. VARIANTS OF PCR-II 1 Lecture- 28

2. Reverse Transcriptase-PCR RT-PCR is a technique used to amplify cDNA copies of RNA . First Strand Reaction RNA strand is first reverse transcribed into a ss cDNA template using dNTPs and an RNA-dependent DNA polymerase (reverse transcriptase) through the process of reverse transcription. An oligodeoxynucleotidyl primer is hybridized to 3'-end of mRNA and is then extended by a reverse transcriptase to make a cDNA copy that can be amplified by PCR. This reaction is usually carried out at 37 o C. Second Strand Reaction After the reverse transcription reaction is complete and a cDNA has been generated from the original ss mRNA, second strand reaction is initiated. In this step, standard PCR is performed in the presence of gene-specific reverse and forward primers.   2

3. Applications of RT-PCR RT-PCR is widely used in the diagnosis of genetic diseases Determination of the abundance of specific different RNA molecules within a cell or tissue as a measure of gene expression. RT-PCR can also be very useful in the insertion of eukaryotic genes into prokaryotes . RT-PCR is commonly used in studying the genomes of viruses whose genomes are composed of RNA, such as Influenza virus A and retroviruses like HIV. 3

4. Band-Stab PCR If during amplification the yield is very low, then the desired fragment can be recovered by gel electrophoresis and reamplified , which is known as band-stab PCR. In band-stab PCR, ethidium bromide ( EtBr ) stained agarose gel is analyzed by UV illumination and excess fluid is removed by placing a piece of Whatman 3 MM paper on the surface of the gel. Each band of interest is sampled carefully with the help of hypodermic needle. The needle is withdrawn, the tip is washed in PCR mixture, and the DNA of that particular band is reamplified by using nested primers. 4

5. Degenerate PCR Degenerate PCR is similar to standard PCR, except, that instead of using specific primers of a given sequence, mixed primers are used in degenerate PCR. This PCR variation has proven to be a very powerful technique to find new genes or gene families. Most of the genes that belong to a gene family share structural and functional homologies. If the sequence for a gene from one organism is known and the gene from another closely related organism is desired, the DNA sequence for the particular gene in both organisms will be close, though rarely similar. ' Clustal ' alignment of the protein sequences from a number of related proteins can be carried out to find the conserved and variable domains. The primers are designed on the basis of conserved protein motifs. 5

6. Anchored PCR In some type of PCR where only enough information to make a single primer is known, a known sequence is added to the end of the DNA by enzymatic addition of a polynucleotide stretch or by ligation of known sequence, and the second primer is designed by sequences of anchored DNA. This technique of amplification with single sided specificity has been known as one-sided PCR or anchored PCR. The anchored or single-sided PCR allows specific amplification of DNA where the 5' sequence of the molecule of interest is unknown. This approach is based on homopolymer tailing of cDNA catalyzed by the terminal deoxynucleotidyl transferase. The amplification is performed by using one primer specific for the molecule of interest (gene-specific primer), and a second primer containing a defined 'anchor' sequence attached to a homopolymer sequence complementary to the tail. 6

7. Ligation-anchored PCR ( LA-PCR) An innovative technique of anchord PCR has been developed in which an anchor of defined sequence is directly ligated to first cDNA strand, and the resulting product is amplified by using primers specific for both the cDNA of' interest and the anchor. 7

8. Applications of LA-PCR The production of general cDNA libraries from very small amounts of starting material The analysis of T-cell receptor and immunoglobulin V regions To clone developmentally regulated mRNAs detected with gene trap vectors To characterize alternative promoter usage and splice products 8

9. Asymmetric PCR A PCR technique in which the predominant product is a ss DNA as a result of unequal primer concentration is known as asymmetric PCR. Asymmetric PCR is carried out as usual, but with a great excess of the primers for the chosen strand. As asymmetric PCR proceeds, the lower concentration primer (P L ) is quantitatively incorporated into ds DNA. The higher concentration primer ( P x ) continues to synthesize DNA, but only of its template strand. This generates one of the strands by linear amplification, and a fraction of its total product as ds DNA, limited by the concentration ratio of the primers used. Thus, single stranded target strand along with the ds DNA can be generated by an asymmetric PCR. 9

10. Differential display Reverse Transcriptase PCR (DDRT-PCR of DD-PCR) This is a technique to compare and identify changes in gene expression at mRNA level between any pair of eukaryotic cell samples. With the help of this technique differentially expressed genes are identified. A limited number of short arbitrary primers in combination with the anchored oligo-dT primers are used. The DDRT-PCR method consists of two major steps: ( i ) Reverse transcription of RNAs isolated from different samples with a set of degenerate, anchored oligo ( dT ) primers to generate cDNA pools (ii) PCR amplification of random partial sequences from the cDNA pools with a limited number of short arbitrary primers. 10

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12. Advantages of DDRT-PCR It can detect changes in expression of closely related genes that is not possible from subtraction libraries. 12

13. Drawbacks of DDRT-PCR It is quite frustrating that only a part of mRNA in a cell can be amplified, because for amplification the template should be hybridized with a particular sense and antisense primer; The amplified product must be of the length of 100 -500 bp so that it can be visualized on gel, i.e., the sense primer should bind at considerable distance from the poly (A) tail; There are chances of obtaining a number of false positives, which cannot be confirmed by blotting; The amplified pattern changes with different preparations of RNAs and different types of Taq DNA polymerases; 13

14. Real-Time PCR Real-time PCR, also known as kinetic PCR, qPCR, qRT -PCR, and RT-qPCR, is a quantitative PCR method for the determination of copy number of PCR templates such as DNA or cDNA in a PCR reaction. Real-time PCR monitors the fluorescence emitted during the reaction as an indicator of amplicon production at each PCR cycle, as opposed to the endpoint detection in standard PCR. The fluorescence emitted acts as an indicator of' the amount of PCR amplification that occurs during each PCR cycle Thus, in real-time PCR machines, one can visually see the progress of the reaction in 'real-time'. It is the method of choice for measuring changes in gene expression. 14

15. The instrumentation platform for real-time PCR consists of: ( i ) Thermal cycler (ii) Computer (iii) Optics for fluorescence excitation and emission (iv) Data acquisition and analysis software. 15

16. Quantitative Real Time (QRT) PCR Position of the steep part of the curve varies depending on the amount of template DNA or RNA 16

17. P rinciple of quantitative real-time PCR … use when rather than how much fluorescent signal  PCR cycles  Low (high copy no.) High C T (low copy no.) real-time PCR amplification plots (7 x 10-fold dilns. + NTC) End point (not quantitative) threshold of detection C T 17

18. Methods for qRT -PCR TaqMan Method A probe is required for specific targeting Fluorescein dye along with quencher is used with probe High specificity SYBR Green Method SYBR Green dye is used for fluorescence No probe is required Low specificity 18

19. Real-time PCR 19

20. TaqMan ® chemistry (fluorogenic 5' nuclease assay) R Q forward primer reverse primer 3' 3' 5' 5' 3' 5' 5' 1. polymerisation probe R = reporter dye Q = quencher dye 3' 5' 5' 3' 5' 5' 4. polymerisation completed Q R 3' R Q 3' 3' 5' 5' 3' 5' 5' 2. strand displacement Q 3' 3' 5' 5' 3' 5' 5' 3. cleavage R 20

21. Taqman Probes probe contains fluorescent tag and quencher exonuclease activity of Taq polymerase releases fluorescent tag high background from probe Primer/Probe Design 50-150 bp ( amplicon ) 20-26 bases (probe) T m of probe 8-10 o > annealing temperature 21

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23. Applications of Real-time PCR Commonly used for both basic research and diagnostics. R eal-time PCR is mainly used to provide quantitative measurements of gene transcriptio n . R eal-time PCR is applied to rapidly detect infectious diseases , cancer and genetic abnormalities. 23