PCR.pptx

pcr, polymerase chain reaction,
Asymmetric PCR
Nested PCR
Multiplex PCR
Competitive PCR
RT-PCR (reverse transcriptase)
Real-time PCR
Hot start PCR
Inverse PCR

1. Polymerase Chain Reaction (PCR) and Its Applications Dr. Dinesh Jain Associate Professor SMS Medical College Jaipur

2. What is PCR? It was invented in 1983 by Dr. Kary Mullis, for which he received the Nobel Prize in Chemistry in 1993. PCR is an exponentially progressing synthesis of the defined target DNA sequences in vitro .

3. What is PCR? : Why “Chain”? It is called “chain” because the products of the first reaction become substrates of the following one, and so on.

4. 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

5. Hot-Start PCR Inverse PCR Multiplex PCR Nested PCR Ligation-Mediated PCR Methylation -Specific PCR (MSP) Multiplex Ligation-Dependent Probe Amplification (MLPA) Thermal Asymmetric Interlaced PCR (Tail- PCR) Assembly PCR Asymmetric PCR Colony PCR Helicase -dependent amplification In Situ PCR (ISH) Intersequence -specific PCR (ISSR) Ligation-mediated PCR Methylation -specific PCR (MSP) Long PCR Miniprimer PCR Overlap-extension PCR Quantitative PCR (Q-PCR) Reverse Transcription PCR (RT-PCR Solid Phase PCR Touchdown PCR (Step-down PCR) Universal Fast Walking Variable Number of Tandem Repeats (VNTR) PCR InterSequence -Specific PCR (or ISSR-PCR) Types of PCR

6. The Reaction THERMOCYCLER PCR tube

7. Applications of PCR Classification of organisms Genotyping Molecular archaeology Mutagenesis Mutation detection Sequencing Cancer research Detection of pathogens DNA fingerprinting Drug discovery Genetic matching Genetic engineering Pre-natal diagnosis

8. Applications of PCR Basic Research Applied Research Genetic matching Detection of pathogens Pre-natal diagnosis DNA fingerprinting Gene therapy Mutation screening Drug discovery Classification of organisms Genotyping Molecular Archaeology Molecular Epidemiology Molecular Ecology Bioinformatics Genomic cloning Site-directed mutagenesis Gene expression studies

9. Applications of PCR Molecular Identification Sequencing Genetic Engineering Molecular Archaeology Molecular Epidemiology Molecular Ecology DNA fingerprinting Classification of organisms Genotyping Pre-natal diagnosis Mutation screening Drug discovery Genetic matching Detection of pathogens Bioinformatics Genomic cloning Human Genome Project Site-directed mutagenesis Gene expression studies

10. Steps in PCR Initialization Denaturation Annealing Extension / Elongation Final elongation Final hold Initialization step Heating the reaction to a temperature of 94-96°C for 1-9 minutes.

11. Denaturation step 94-98°C for 20-30 seconds . Denaturation of DNA template by disrupting the hydrogen bonds between complementary bases of the DNA strands, yielding single strands of DNA .

12. Annealing step 50-65°C for 20-40 seconds Stable DNA-DNA hydrogen bonds are formed The polymerase binds to the primer-template hybrid and begins DNA synthesis.

13. Extension/elongation step 75-80°C At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template by adding dNTPs in 5' to 3' direction.

14. Final elongation 70-74°C for 5-15 minutes To ensure that any remaining single-stranded DNA is fully extended. Final hold 4-15°C for an indefinite time short-term storage of the reaction

15. Allele- Specific PCR Selective PCR amplification of the alleles to detect single nucleotide polymorphism (SNP) Selective amplification is usually achieved by designing a primer such that the primer will match or mismatch one of the alleles at the 3’ end of the primer.

16. Asymmetric PCR It is used for DNA sequencing The two primers are used in the 100:1 ratio so that after 20-25 cycles of amplification one primer is exhausted thus single stranded DNA is produced in the next 5-10 cycles

17. Real Time PCR Quantitative real time PCR (Q-RT PCR) It is used to amplify and simultaneously quantify a target target DNA molecule Real time PCR using DNA dyes Fluorescent reporter probe method

18. Real Time PCR

19. Helicase-dependent amplification  Constant temperature is used rather than cycling through denaturation and annealing/extension cycles.  DNA Helicase , an enzyme that unwinds DNA, is used in place of thermal denaturation.

20. Intersequence-specific PCR (ISSR): A PCR method for DNA fingerprinting that amplifies regions between some simple sequence repeats to produce a unique fingerprint of amplified fragment lengths.

21. Inverse PCR  A method used to allow PCR when only one internal sequence is known.  This is especially useful in identifying flanking sequences of various genomic inserts.

22. Anchored PCR When sequence of only one end of the desired segment of gene is known,the primer complimentary to the 3' strand of this end is used to produce several copies of only one strand of the gene.

23. RT-PCR ( Reverse Transcription PCR)  It is used to amplify, isolate or identify a known sequence from a cellular or tissue RNA .  RT-PCR is widely used in expression profiling , to determine the expression of a gene or to identify the sequence of an RNA transcript. RACE-PCR  Used to obtain 3' and 5' end sequence of cDNA transcripts

24.   Parameter PCR Gene cloning 1. Final result Selective amplification of specific sequence Selective amplification of specific sequence 2. Manipulation In vitro In vitro and in vivo 3. Selectivity of the specific segment from complex DNA First step Last step 4. Quantity of starting material Nanogram (ng) Microgram (m) 5. Biological reagents required DNA polymerase (Taq polymerase) Restriction enzymes, Ligase, vector. bacteria 6. Automation Yes No 7. Labour intensive No Yes 8. Error probability Less More 9. Applications More Less 10. Cost Less More 11. User’s skill Not required Required 12. Time for a typical experiment Four hours Two to four days Comparison PCR - Polymerase Chain Reaction and Gene Cloning

25. Application of PCR Cloning a Gene encoding a known protein Amplification of old DNA Amplifying cloned DNA from Vectors Rapid Amplification of cDNA ends Detecting Bacterial or Viral Infection ● AIDS infection ● Tuberculosis (Mycobacterium tuberculosis)

26. Genetics Diagnosis Diagnosing inherited disorders Cystic fibrosis Muscular dystrophy Haemophilia A and B Sickle cell anaemia Diagnosing cancer Blood group typing.

27. Problems with PCR Polymerase errors Polymerase lacks exonuclease activity Size limitations PCR works readily with DNA of lengths two to three thousand basepairs Non specific priming

28. RT-PCR The enzyme reverse transcriptase is used to make a DNA copy (cDNA) of an RNA template from a virus or from mRNA. Normal PCR with two primers

29. Multiplex PCR Use of multiple sets of primers to detect more than one organism or to detect multiple genes in one organism. Remember, the PCR reaction is inherently biased depending on the G+C content of the target and primer DNA. So performing multiplex PCR can be tricky . or

30. Seminested PCR Three primers are required, the normal upstream and downstream primers as well as a third, internal primer. Two rounds of PCR are performed, a normal PCR with the upstream and downstream primer, and then a second round of PCR with the downstream and internal primer. A second smaller product is the result of the second round of PCR.

31. ICC-PCR Integrated cell culture PCR is used for virus detection. Cell culture takes 10 – 15 days. PCR alone detects both infectious and noninfectious particles. So use a combination of these techniques: grow the sample in cell culture 2 – 3 days, release virus from cells and perform PCR. This results in the detection of infectious virus in a shorter time with a 50% cost savings. It also allows use of dilute samples which reduces PCR inhibitory substances.

32. Labelling approaches CYBR green Real-Time PCR This technique allows quantitation of DNA and RNA. Reactions are characterized by the point in time during cycling when amplification of a PCR product is first detected rather than the amount of PCR product accumulated after a fixed number of cycles. The higher the starting copy number of the nucleic acid target, the sooner a significant increase in fluorescence is observed. TAQ-man probes FRET probes

33. PCR fingerprinting AP-PCR (arbitrarily primed PCR), 1 primer required, 10-20 bp, no sequence information required REP-PCR (repetitive extragenic palindromic sequences) 2 primers insert randomly into the REP sites ERIC-PCR (enterobacterial repetitive intergenic consensus sequences), 2 primers insert randomly into the ERIC sites, best for Gram Negative microbes All of these fingerprinting techniques tell one if two isolates are the same or different. They do not provide information about the identity or relatedness of the organisms

34. RT-PCR lab You have a cell…is a certain gene on (by “on,” we mean active and producing mRNA?)? If a certain gene is on when the cell divides, the gene might produce a protein that causes cell division….

35. Central Dogma: DNA has genes and is in nucleus TRANSCRIPTION : Double strands of DNA unwind to allow synthesis of messenger RNA (mRNA) from one strand (the coding strand) The mRNA moves out of the nucleus to the cytoplasm mRNA binds to Ribosomes to code for a protein- protein made (translation) Protein carries out intent of gene (red hair protein = hair gene)

36. DNA Structure

37. Unwind, mRNA is made off DNA template- similar to this picture of DNA made off of DNA. Nucleotides pair up: G always pairs with C, T pairs with A. Except in RNA, T is replaced with U.

38. Transcription: RNA synthesis (note coding and template strands) (ch.21)

39. Making mRNA off DNA:

40. So, first step of RT PCR is: ISOLATE THE mRNA from the cell Next, make DNA from the mRNA This is reversing “transcription”– so use an enzyme originally obtained from viruses– ENZYME IS CALLED REVERSE TRANSCRIPTASE (THE RT OF RT PCR)

41. Last slide: this is the RT part of RT PCR PCR part: After RT, you now have a tiny, trace amount of what is called complimentary DNA (cDNA). This tiny trace amount is not enough to sequence. Next, you have to make enough copies of the tiny trace amount of cDNA to sequence