Lecture 7 - PCR

Polymerase chain reaction (PCR)

  • Rapid, repetitive copying of a section of double-stranded DNA located between two regions of a known sequence

  • Allows for rapid replication of a single copy of DNA

  • Requires two specific primers that bind to both DNA strands in opposing directions

Method

  1. Heat denaturation at 95 degrees

  2. Annealing at 40-65 degrees → primers bind to complementary sequences

  3. Extension at 72 degrees → DNA polymerase synthesise new strands starting from annealed primers

  4. Repeat steps 1,2,3 for multiple cycles

  5. Double stranded PCR products covering the region we want to amplify are formed exponentially whilst products containing one strand DNA of an undetermined length are formed arithmetically

  6. After 25-35 cycles, a discrete DNA product is formed that can be visualised by Agarose Gel Electrophoresis

  • Temperature changes in seconds

  • Heat transfer is very efficient

  • Heated lid prevents evaporation of sample during high temperature steps

PCR Contains:

  • Template

  • DNA polymerase

  • Primers

  • Reaction Buffer

  • dNTPs (ATP, CTP, GTP, TTP)

Template

Wide range of DNA molecules → genomic DNA, plasmids, fragments, phages, previously amplified fragments

Template concentration → sensitive enough to amplify 10 copies of target sequence (from 10-6 μg of DNA)

Depends on template complexity:

  • Genomic DNA → Use high template concentration

  • Plasmid DNA → Use low template concentration

Too much template yields non-specific products whilst low template results in low yield

Quality of template → avoid contamination with DNA polymerase inhibitors like proteases, chelating agents, detergents and organic solvents

DNA polymerase

Thermostable → does not denature at high temperatures used during denaturing steps and isolated from thermophilic microorganisms eg. Thermus aquaticus, Pyrococcus furiosus

Proofreading activity:

  • 3’ → 5’ exonuclease activity allows for removal of mismatched nucleotides reducing frequency of erros

  • Taq polymerase → lacks 3’ → 5’ proofreading, used in routine detection PCR

  • Pfu polymerase → has 3’ → 5’ proofreading activity, useful when PCR product will be used in cloning or gene expression experiments

Types of ends generated after PCR:

  • Polymerases without proofreading activity add an extra base to the ends of the PCR product, typically an A

  • Polymerases with proofreading activity generate blunt-ended PCR products that can be ligated to any blunt ended site on a vector

Primers

  • Single stranded DNA oligonucleotides that are complementary to regions flanking the sequence of interest

  • 15-30 bases in length, should contain 40-60% GC content

  • Sense Primer sequence is the same as the Sense strand and is complementary to Anti-Sense strand

  • Anti-sense primer sequence is the same as Anti-sense strand and is complementary to the Sense strand

Additional Considerations in Primer Design

  • Avoid sequences that form hairpin loops

  • Avoid sequences that bind to each other (primer dimers) or repetitive sequences

  • Primer requires a perfect match at the 3’ end so avoid mismatches at the 3’ end of the primer

Annealing Temperature

  • Temperature at which the primer binds in a stable manner to the template

  • Usually is 2-5 degrees below melting temperature of the primer-template hybrid but it must be determined experimentally

  • Tm = [(A+T) x 2 + (G+C) x4]

  • The Tm of both primers should be similar

  • If primers have different Tm then determine annealing temperature from lower Tm

  • When annealing temperature is too low, the primer may bind to non-specific templates and the amplification of wrong products can occur

  • This can also occur when primer concentration is too high

Primer modifications

  • Primers are incorporated into the PCR product sequence so specific sequences can be added to the PCR product by modifying the 5’ end of the primer

  • You can add a restriction site to primer so that all new PCR products have restriction sites so they are easy to clone

Cloning PCR products

  • DNA polymerase lacking proofreading activity (Taq) adds an A overhang to the 3’ end of PCR products

  • There are commercially available vectors with a 3’ T overhang

  • Blue-White selection for insert

  • Convenient restriction sites flanking insert to facilitate further sub-cloning steps

PCR reaction conditions

Standard Components in a PCR Buffer

  • 20mM Tris/HCl → pH 3.8

  • 10mM KCl

  • 2mM MgCl2

  • K+ and Mg2+ bind to the phosphate backbone (negative charge) and contribute to stabilising the primer annealing to the template

  • Mg2+ is also needed for DNA polymerase activity and fidelity of replication

  • If Mg+ concentration is too high non-specific annealing and DNA amplification may occur, leading to amplification of non-specific products

PCR applications

  •  Preparing probes for Hybridisation experiments 

  • Assays for the presence of Infectious agents

  • Direct Cloning from genomic DNA or cDNA

  • Quantification of rare or low abundance DNA and RNA 

  • In vitro mutagenesis

  • DNA cycle sequencing

  • Genetic Fingerprinting of Forensic samples

  • Analysis of allelic sequence variations

  • Prenatal Diagnosis of genetic diseases

PCR and Genetic Disease

  • PCR allows for rapid genotyping of genetic markers

  • Used to detect deletion/insertion or point mutations

Microsatellites

Microsatellites → short tandem repeated DNA segments, most are bordered by unique DNA sequences

Huntington Disease:

  • Caused by dominant mutant allele of HD gene, encoding the protein Huntingtin

  • The mutation is a CAG trinucleotide repeat in exon 1

  • Healthy allele contains 10-35 CAG repeats and mutant allele has 36-121 repeats

  • Variations in the number of tandem repeats results in different sized PCR products that can be detected by gel electrophoresis

Sickle cell anaemia

  • Single base pair (A to T) mutation in β-globing gene

  • In homozygous individuals abnormal haemoglobin is produced

  • The mutation causing sickle cell anaemia occurs in exon 1

  • The normal codon is GAG (glutamic acid), while the mutant codon is GTG (valine)

  • Primer 1 (red) and Primer 2 (blue) are designed to flank the region containing the mutation

  • PCR amplifies this short region of DNA from the patient’s genome.

  • Both the normal and mutant alleles can be amplified

  • After amplification, the sequence is tested for the A→T mutation.

  • In this example, allele-specific PCR is used:

    • One primer matches the normal allele (GAG)

    • Another matches the mutant allele (GTG)

Molecular Epidemiology of Tuberculosis

  • Mycobacterium tuberculosis genome contains several variable-number tandem repeats units called Mycobacterial Interspersed Repetitive Units (MIRUs)

  • PCR amplification of MIRUs categorises the number and size of each repetitive unit 

  • This permits genotyping comparison of strain X and type strains