chapter 14: dna amplification by polymerase chain reaction

Chapter 14: DNA Amplification by Polymerase Chain Reaction

Polymerase Chain Reaction (PCR) Copies (“amplifies”)DNA in a test tube

using the same type of chemistry that cells use to copy DNA Exponential amplification of specific, short

(usually 2,000 bp or less) sequences of DNA Products are called amplicons

Highly sensitive Can amplify small quantities Rapid and robust

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Reaction ingredients: PCR Primers

▪ Short, single-stranded DNA polynucleotides that are complementary to the sequences which flank the target region

dNTPs (in abundance) Template DNA DNA Polymerase

▪ Thermostable (e.g. Taq polymerase) MgCl2 and buffer

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PCR steps: Denaturation (94 deg C) Annealing (typically 50-60 deg C)

▪ Set just below melting temperature of primers▪ 4 + 2 rule

Extension (72 deg C)▪ Optimum temp for taq polymerase

Cycling (denaturation, annealing, extension)▪ Typically 20-30 times

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5

1 copy

2 copies

Repeat 30 times = 230 = 1 billion copies

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PCR controls Used to monitor effectiveness Positive control

▪ Indicates that all reaction ingredients are working

▪ Known sample Negative control

▪ Indicates that there is no contaminating DNA in any of the reaction reagents

▪ Includes all reaction ingredients except template DNA

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Template DNA degradationLow copy number of template

Stochastic EffectPCR inhibitors

Heme, Indigo dyes, Melanin from hairsContamination

Pre-PCR and post-PCR should be in separate areas

Supplies and reagents also separated8

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Activity Cellular Replication

PCR

Denaturation

Primers

Extension

Number of copies produced

Size of region copied

Ingredients

Purpose