PCR. CSIBD Molecular Genetics Course July 12, 2011 Michael Choi, M.D.

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1 PCR CSIBD Molecular Genetics Course July 12, 2011 Michael Choi, M.D.

2 General Outline of the Lecture I. Background II. Basic Principles III. Detection and Analysis of PCR Products IV. Common Applications of PCR V. Real-time PCR

3 The Nobel Prize in Chemistry 1993 Decisive progress in gene technology through two new methods: the polymerase chain reaction (PCR) method and site-directed mutagenesis Press Release 13 October 1993 The Royal Swedish Academy of Sciences has decided to award the 1993 Nobel Prize in Chemistry for contributions to the development of methods within DNA-based chemistry, with half to Dr Kary B. Mullis, La Jolla, California, U.S.A., for his invention of the polymerase chain reaction (PCR) method, and half to Professor Michael Smith, University of British Columbia, Vancouver, Canada, for his fundamental contributions to the establishment of oligonucleotide-based, site-directed mutagenesis and its development for protein studies.

4 II. Basic Principles: PCR cycle design - three steps DNA denaturation (typically C): Separates strands of substrate DNA for each cycle Complete denaturation essential to efficient PCR Typically 30 sec to 2 min Primer Annealing (typically C): Allows primer binding to target DNA sequences Typically 30 sec to 2 min DNA Extension (typically C): Generates DNA product by extending hybridized primer 1 min generally adequate for product up to 1-2 kbp

6 II. Basic Principles : Reaction Components Primers (amplimers): sequences may be precise, degenerate or nonspecific, depending on application; generally requires some sequence information Target DNA: routine PCR excellent for amplification of nucleotide sequences; long-range PCR capable of amplifying at least 10kb Thermostable DNA polymerase: Taq notable for high processivity, lack of proofreading (3'-5' exonuclease activity) Pfu notable for proofreading activity but decreased efficiency dntps: Best to use equal concentrations to minimize misincorporation Divalent cations (MgCl2): Concentration affects annealing, denaturation, primer-primer interactions, efficiency of polymerase 2 mm generally a good starting point, but may need to test varying concentrations (e.g., mM) to optimize reaction

II. Basic Principles: PCR primer design Usually 18-25 nt Primer 3: http://frodo.wi.mit.

7 II. Basic Principles: PCR primer design Usually nt Primer 3: GC content should be between 40-60% Melting temp (Tm) of two primers should not differ by more than 5 degrees Inverted repeats or any selfcomplementary sequences >3bp should be avoided The 3 sequence of one primer should not be complementary to any region of the other primer in the same direction (avoid primer dimers)

8 Primer Dimers

9 II. Basic Principles: Pros and Cons of PCR Pros Speedy and easy Sensitivity: theoretically only one molecule of template needed great for biomedical research, forensics, molecular anthropology, etc. Robustness: often possible to amplify DNA from tissues or cells which are badly degraded or embedded Cons Need for target sequence info Short size and limited amounts of product: typically in 0-5 kb range but long range PCR protocols have been developed up to tens of kbs Infidelity of DNA replication: Taq: 1 error/10,000 bases/cycle; Pfu: has 3'-5' exonuclease proofreading activity but less efficient Contamination: aliquots, gloves, no talking, good pipetting technique

10 III. Detection and Analysis of PCR Products A. Gel Electrophoresis 1.Ethidium bromide staining 2.Autoradiography of labeled products 3.Direct or indirect nonisotopic labeling (e.g., fluorescent) 4.Blot hybridization (Southern or dot blot) B. Sequencing of PCR products 1.Direct sequencing avoids need to clone products

11 IV. Common Applications of PCR: Cloning of PCR products in bacterial cells

12 IV. Common Applications of PCR: Restriction site polymorphisms can easily be typed by PCR as an alternative to laborious RFLP assays

13 IV. Common Applications of PCR: Correct base-pairing at the 3 end of PCR primers is the basis of allele-specific PCR

14 IV. Common Applications of PCR: PCR can be used to type short tandem repeat polymorphisms (STRPs)

15 IV. Common Applications of PCR: Example of typing for a CA repeat Genotypes 1(3,6) 2(1,5) 3(3,5) 4(2,5) 5(3,6) 6(2,5) 7(3,5) 8(3,6).. Slipped strand mispairing is thought to be responsible for shadow bands in tandem nucleotide repeats: Strong main band followed by two lower shadow bands

16 IV. Common Applications of PCR: Linker-primed PCR permits indiscriminate amplification of DNA sequences in a complex target DNA

17 IV. Common Applications of PCR: Dideoxy DNA sequencing relies on synthesizing new DNA strands from a singlestranded DNA template and random incorporation of a base-specific dideoxynucleotide to terminate chain synthesis

18 Structure of a dideoxynucleotide, 2, 3 dideoxy CTP

19 IV. Common Applications of PCR: Automated DNA sequencing using fluorescent primers

20 IV. Common Applications of PCR: PCR mutagenesis

21 V. Real-time PCR Normal reverse transcriptase PCR is only semiquantitative at best because, in part, of the insensitivity of ethidium bromide. Thus real time PCR was developed because of: The need to quantitate differences in mrna expression The availability of only small amounts of mrna in some procedures such as in the use of: --- cells obtained by laser capture micro-dissection --- small amounts of tissue --- primary cells --- precious reagents

V. Real-time PCR Quantitation of the amount of cdna in the original sample must be done where the amplification is exponential and, this is at the very beginning of the upturn of the curve.

22 V. Real-time PCR Quantitation of the amount of cdna in the original sample must be done where the amplification is exponential and, this is at the very beginning of the upturn of the curve. In real time PCR, we measure the cycle number at which the increase in fluorescence (and therefore cdna) is exponential. This is shown by the orange horizontal line in the figure and is set by the user. The point at which the fluorescence crosses the threshold is called the Ct

BIOLOGY Dr.Locke Lecture# 27 An Introduction to Polymerase Chain Reaction (PCR)

BIOLOGY Dr.Locke Lecture# 27 An Introduction to Polymerase Chain Reaction (PCR) BIOLOGY 207 - Dr.Locke Lecture# 27 An Introduction to Polymerase Chain Reaction (PCR) Required readings and problems: Reading: Open Genetics, Chapter 8.1 Problems: Chapter 8 Optional Griffiths (2008) 9