The Polymerase Chain Reaction. Chapter 6: Background

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1 The Polymerase Chain Reaction Chapter 6: Background

2 PCR Amplify= Polymerase Chain Reaction (PCR) Invented in 1984 Applications

3 Invention of PCR Kary Mullis Mile marker in April of 1983 Pulled off the road and outlined a way to conduct DNA replication in a tube

4 DNA Replication vs. PCR PCR The laboratory version

5 What is PCR? Polymerase chain reaction or PCR Simplified version Copies a specific sequence of DNA Target is Annealing

6 PCR: the in vitro version of DNA Replication The following components are needed to perform PCR in the laboratory: 1) Template DNA 2) DNA Polymerase 3) Four 4) Buffers 5) Magnesium chloride (MgCl2) 6) Primers 7) Tubes 8) Thermal cycler

7 Thermal Cyclers Water baths Thermal cyclers

8 Challenges of DNA Replication in a Tube Normal replication: Heat Heat-stable DNA polymerases

9 Heat-stable DNA Polymerase PCR involves very high temperatures DNA polymerases would denature Taq DNA polymerase Taq

10 The three main steps of PCR Basis of PCR: In a PCR reaction: Step 1: Denature DNA Step 2: Primers Anneal Step 3: DNA polymerase Extends the DNA chain

11 Denaturation of DNA

12 Step 2 Annealing or Primers Binding Reverse Primer Forward Primer

13 Step 3 Extension or Primer Extension extension extension

14

15 The Size of the DNA Fragment Produced in PCR is Dependent on the Primers The PCR reaction Forward primer Reverse primer

16 The DNA of interest is amplified by a Example: power of 2 for each PCR cycle

17

18 Overview of PCR 1. Temperature Cycling Denaturation Annealing Extension 2. Every cycle DNA between primers is duplicated

19 PCR Amplification

20 Types of PCR There are many variations of traditional PCR Real-time or quantitative PCR (qpcr) Reverse transcription PCR (RT-PCR) Multiplex PCR Degenerate PCR Nested PCR Random amplification of polymorphic DNA (RAPD) Fast PCR

21 PCR Optimization Each reaction is very different Factors that must be taken into account vary with the purpose of the reaction

22 PCR Optimization Conditions and factors to optimize:

23 PCR Optimization Template quality

24 PCR Optimization Template concentration

25 PCR Optimization Primers Primers bind to their complementary sequence on the target DNA

26 PCR Optimization Primer concentration The normal range: Increasing the concentration can lead to

27 PCR Optimization Primer design The sequence of the primers is crucial to the success of PCR T m Annealing temperature

28 PCR Optimization Magnesium concentration Magnesium Normal concentration

29 PCR Optimization Annealing temperature Annealing temperature: Annealing temperatures are often adjusted

30 PCR Optimization Cycling temperatures and times Amount of time that each step Only the annealing temperature is adjusted

31 PCR Optimization

32 PCR has become a very powerful tool in molecular biology Start with a single sperm cell or stand of hair Can amplify fragments of interest Can use the selectivity of the primers

33 Other Applications of PCR PCR in medicine

34 PCR and prenatal diagnosis For prenatal diagnosis, PCR used to amplify DNA Single base changes then detected by: - - Combination of two methods provides confirmation.

35 Other Applications of PCR PCR in medicine Detection of disease-causing organisms Amplification of disease-causing organisms for quick diagnosis Amplification of genes of interest for cloning

36 Other Applications of PCR PCR and Disease Primers can be created that will only bind and amplify certain alleles of genes or mutations of genes Some diseases that can be diagnosed with the help of PCR:

37 Huntington s Disease (HD) HD Caused by: In individuals with HD, the HD gene is expanded In non-hd individuals: In HD individuals: PCR can be performed on an individual s DNA to determine whether the individual has HD.

38 Cystic Fibrosis (CF) CF Caused by: In non-cf individuals: In CF individuals, The presence of CTFR mutations in a individual can be detected by performing PCR and sequencing on that individual s DNA.

39 Human Immunodeficiency Virus (HIV) HIV HIV tests PCR product = No PCR product = Before the appearance of HIV antibodies PCR can detect:

40 Other Applications of PCR PCR in agriculture GMO Compliance with reporting requirements

41 PCR in Forensics Crucial forensic evidence may be present in very small quantities. Too little material PCR also possible on extensively degraded DNA. Other advantages of PCR in forensic science are: Major legal problems with PCR:

42 Other Applications of PCR PCR in forensics Short Tandem Repeat (STR) analysis is commonly used in forensics PCR used: Regions contain Primers are chosen that will amplify these repeated areas

43 Other Applications of PCR PCR in forensics Short tandem repeats (STR) analysis Short tandem repeats are amplified and used as a method of identification

44 Using STR to compare forensic and suspect samples Individuals A & C are excluded by this analysis. The samples from individual B will be subjected to further tests.

45 Other Applications of PCR PCR in forensics Allele frequencies Combined DNA Index System (CODIS)

46 Other Applications of PCR PCR in paternity Samples are amplified using STR analysis

47 Other Applications of PCR PCR in human migration Short interspersed repetitive elements (SINEs) have been inserted randomly into our human genome over millions of years.

48 Other Applications of PCR PCR in wildlife conservation Poaching endangers many animals in the world today

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