Polymerase Chain Reaction (PCR) Made Easy

What is Polymerase Chain Reaction ?

It is a process of artificial DNA replication (amplification of a selected part of DNA). As the name suggests:

1. Polymerase: It requires DNA polymerase for extending the added primers to complete DNA replication.
2. Chain Reaction: PCR is run in cycles. The number of molecules increase exponentially as the cycle is repeated. Example:
   • We have 1 original molecule of DNA at the beginning
   • After 1st cycle: We have 2 copies of the molecule
   • After 2nd cycle: We have 4 copies of the molecule
   • After 3rd cycle: We have 8 copies of the molecule and so on….

Components Needed for PCR

As PCR is also called as “molecular photocopying”, we use the analogy of a Photocopy to learn the role of components of PCR.

A page to be photocopied is the Target Sequence – It contains important information of our interest among the whole book (DNA template). It may be containing a suspected mutation, short tandem repeat (STR), etc.

A bookmark identifies the specific page to photocopy out of a book – PCR primers identify the specific fragment to be copied from the entire genome.

To copy a page, the photocopier uses the paper and toner to make the copy – the DNA polymerase requires nucleotides to produce a replicate of the original DNA fragment.

Steps of PCR

1. Solution containing components of PCR (explianed above) prepared.

2. Denaturation of dsDNA by heating to 95°c for few minutes (in vivo, this would be mediated by helicase).

3. Annealing of DNA primer specific for region of interest and slowly cooling down the solution to 40°c (in vivo, primer would be synthesize by the enzyme primase – but in vitro as in PCR, we need to add them externally).

• The primers are 2 synthetic oligonucleiotides: one is complementary to a short sequence in one strand of the DNA to be amplified, and the other is complementary to a sequence in the other DNA strand.
• Primers provide 3′-OH end for extending the newly synthesized DNA strand.
• Example: To amplify the CAG repeat in …CTC AAG TCC TTC [CAG]n CAA CAG CCG CCA…
  • This represents 5′ → 3′. Remember that it also has complementary bases running from 3′ → 5′.
  • So the 2 sets of RNA primers required  containes complementary bases to separated DNA template but runs in 5′ → 3′ direction starting from 3′ end of the template.
  • So, the 2 sets of primers required in this case are:
    • For DNA template running 5′ → 3′: TGGCGGTGTTG
    • For complementary DNA template running 3′ → 5′: CTCAAGTCCTTC

4. Replication of DNA at primer by heat stable DNA polymerase (optimum temperature 72°c i.e. reheating to increase the activity of Taq DNA polymerase).

5. Repetition of process (chain reaction): Generally PCR is run for 20-30 cycles.

Clinical Uses of PCR

1. Diagnosis of viral infection and monitoring of antiviral therapy – HIV, Hepatitis B Virus, Human Cytomegalovirus, etc.

• PCR gives information on viral load.
• In HIV, PCR can be used when measurement of antibody is not reliable:
  • Earlier diagnosis of disease
  • Immune system not competent enough to make antibodies
  • Neonate born to HIV positive mother – even non-infected newborns would have antibodies (transplacental)

2. Diagnosis of bacterial infection:

• Mycobacterium tuberculosis, Chlamydia Trachomatis, Neisseria gonorrheae and Bordetella pertussis.
• Tests based on molecular methods have the advantage of avoiding days or weeks of delay and allow early recognition and treatment.

3. Forensic/Paternity testing:

• use of Variable Number (VNTR) or Short Tandem Repeats (STR) – these are unique copies of non-coding region of DNA between individuals.
• since they exist on both chromosomes, individuals have two copies at each locus – 1 paternal and 1 maternal.
• can only prove with certainty that the sample DOES NOT belong to the test subject – cannot prove with 100% certainty that DNA belongs to individual of interest because there is a small chance that someone shares the same VNTR or STR

4. Direct mutation testing:

• PCR can amplify the region for sequencing

Disadvantage of PCR

A potential problem for PCR is obtaining a pure sample of DNA to start with; any contaminant DNA will also be amplified.

RT-PCR

PCR can also be used to study pattern of gene expression by a process called reverse PCR.

• mRNA is converted into cDNA (copy DNA) by using reverse transcriptase (RNA dependent DNA polymerase)
• CDNA serves as a template for usual PCR procedure
• Clinical use: HIV viral load