PCR Tutorial

 

1.) DNA STRUCTURE

DNA is the genetic code of life. It is the blueprint for organisms - all the information which is needed to build and maintain us is stored in our DNA.

DNA is made up of two strands which are twisted to form a "double helix". The helix is tightly coiled to fit inside the nucleus, the "control room", of our cells. If the DNA inside a human cell was uncoiled it would be approximately 2 metres long.

DNA stands for Deoxyribonucleic Acid.


If you zoom down and look closely at the structure of DNA it will look like a ladder with upright supports and rungs.

The upright supports are made of alternating sugar molecules and phosphates. Attached to each sugar is another component of DNA known as a base.

 
There are only four types of bases found in DNA - Adenine, Guanine, Thymine and Cytosine.

It is the order, or sequence, of the bases which encodes our genes.

When two strands of DNA are facing one another the bases from each strand bond with those opposite, forming the rungs of the ladder. So each rung consists of two bases bonded together.

The DNA from a single human cell contains a total of 3 billion base pairs.


There are only two types of rungs in the DNA ladder. This is because each type of base will only bond with another specific base to form a complementary pair.

Adenine bonds with Thymine.

Cytosine bonds with Guanine.

Although they bond, the attachment between these base pairs is relatively weak and does not withstand high temperatures.



2.)
DENATURATION

The aim of the Polymerase Chain Reaction (PCR) is to identify a particular sequence of DNA and produce many copies of it, until there are so many that they are easier to measure. To do this PCR imitates nature’s way of copying DNA.


The first stage of PCR is to "unzip" the double stranded DNA to produce two complementary strands. Known as denaturation.

This is done by heating the DNA above 90oC, which breaks the weak bonds between the bases of the two strands but leaves the rest of the ladder structure unaffected.


3.) ANNEALING

The single strands of DNA have unpaired bases which are free to bind to new matching bases in the PCR solution. Binding will not happen spontaneously - a primer is needed to start the process.

Primers, small sequences of DNA complementary to the target DNA sequences which need to be copied, are included in the PCR solution. Primers will only bond to their matching target sequence. If no sequences match, PCR will not occur and a negative result will be recorded.

Amplicor primers are approximately 20-30 bases in length.

In the second stage of PCR, the primers bind (known as annealing) to the matching, target sequence of bases on the original DNA.



4.)
EXTENSION

Once the primer has attached to the original DNA, new bases will begin to bind to the primer and extend the DNA strand - this is known as extension.

An enzyme, a protein which promotes a particular chemical reaction, is needed to assist in this process. In PCR, an enzyme called Taq DNA Polymerase promotes extension of the DNA; helping to bind complementary bases to the original DNA which acts like a template.

The bases already have sugar and phosphate groups attached to them, so as they bind to the DNA a new genetic ladder is built.

This continues until a complete copy of the target sequence has been produced, resulting in two double stranded DNA molecules from one original. This is one PCR cycle.

 


5.) PCR CYCLES

The three stage PCR cycle (denaturation, annealing and extension) takes approximately two-three minutes, depending on the length of the target DNA, each cycle increasing the number of DNA copies produced. As PCR continues, first two, then four, then eight copies are created and so on... Each cycle doubles the amount of target DNA - this is the chain reaction in PCR.

The process of PCR is also called amplification because the target sequence is being copied and each copy in turn is being copied over and over again in order to make millions of copies.

In theory, at maximum efficiency, 20 PCR cycles yield a million copies and 30 cycles yield a billion copies. Following PCR there is enough material to detect the target DNA.

The DNA copies are measured by using an enzyme which has previously been bound to the primer used in the PCR reaction. When a particular substance is added to the solution containing the PCR copies, this enzyme causes a chemical reaction which changes the colour of the solution. The intensity of the colour is related to the amount of enzyme present and therefore the number of DNA copies present. By using a machine which measures colour intensity the number of DNA copies and the amount of the original sequence present can then be calculated.

 

 

6.) PCR for HIV RNA

By targeting a specific sequence of HIV’s genetic code, PCR can be used to amplify and then measure the amount of HIV genetic code in the plasma of an individual, which is characteristic of the amount of virus in an individual’s blood.

HIV’s genetic code is present as RNA - Ribonucleic Acid. This is similar to DNA but there are some differences. RNA, for instance, can be single stranded, has a different sugar in its ladder structure and contains the base Uracil instead of Thymine.

Before PCR can begin, the viral RNA must be converted to a complementary strand of DNA. This is know as reverse transcription (RT).



7.)
HIV: RNA to DNA

The single strand of HIV RNA has unpaired bases which are free to bind to new matching bases. As before, this will not happen spontaneously and a primer must start the process. A small sequence of single stranded DNA, which is specific to a target sequence of the viral RNA, is included in the PCR solution.

Once the primer has bound to the viral RNA, new bases will begin to bind to the primer and extend the new DNA strand. An enzyme is needed to assist in this binding process, in this case rTth DNA Polymerase.

As before, the bases being bound to the DNA primer already have sugar and phosphate groups attached to them, so a new ladder is built as new bases are added on.

This continues until a new DNA strand has been produced. This results in a double stranded structure combining one strand of viral RNA and one strand of complementary DNA (cDNA).



8.)
HIV: DENATURATION

Once DNA is present the PCR process can begin. The new combined RNA / cDNA strand is "unzipped" by heat revealing the DNA bases ready for pairing.

 

 

9-11.) HIV: ANNEALING AND EXTENSION

DNA primer, complementary to the target sequence of cDNA, is contained in the PCR solution. Once the primer has bound to the cDNA, new bases will begin to bind and extend the DNA strand.

In PCR involving HIV, the enzyme rTth DNA Polymerase also promotes extension of the DNA.

This continues until a new target sequence has been produced, resulting in two double stranded DNA molecules from one original.

Microwell Plate

The AMPLICOR HIV-1 MONITOR™ is a semi automated system which includes the HIV PCR process. From a small amount of viral RNA extracted from plasma, the AMPLICOR® system will perform reverse transcription and PCR, measure the number of copies of the viral genetic code produced and calculate the original amount of RNA present ie. the amount of viral RNA, and therefore virus, in the plasma.

COBAS AMPLICOR™

Roche Diagnostics is currently developing a fully automated HIV viral load test. The new systems will fully automate all the steps of measuring the HIV viral load in a sample. This will mean that more tests can be carried out at the same time.

For further information on viral load results please click here.