Polymerase Chain Reaction

The polymerase chain reaction (PCR) is a test tube method used to amplify a selected DNA sequence without biologic cloning. The main advantages of PCR over cloning are:

1. It permits the synthesis of millions of copies of a specific nucleotide sequence in a relatively short time (a few hours).

2. It can amplify a DNA sequence, even if its concentration in a mixture does not exceed one part in a million of the total initial sample (i.e. highly sensitive)

3. The method can be used to amplify DNA sequences from any source—bacterial, viral, plant, or animal.

Steps of PCRSteps of PCR 1. The first step of PCR is to denature the DNA,

where the DNA to be amplified is heated by rising the temperature to 92 cº to separate the double-stranded target DNA into single strands.

2. Annealing of “primers“primers” to ssDNA: First of all the sequences of target DNA are not

mandatory to be known. It is only necessary to know the sequences of short segments each side of the target region. These short stretches that bracket the target DNA are known as “the flanking regions”.

Flanking areas act as templates to synthesize two single stranded oligonucleotides (20- 35 bases long) which are complementary to the respective flanking sequences. These synthetic oligonucleotides function as primersprimers in PCR reactions. The 3′-hydroxyl end of each primer points toward the target sequence. The separated strands are cooled (down to 60 cº ) and allowed to anneal to the two primers (one primer for each strand).

3. Chain extension: Both DNA polymerase and deoxyribonucleoside

triphosphates (in excess) are added to the mixture to initiate the synthesis of two new strands complementary to the original DNA chains. DNA polymerase adds nucleotides to the 3′-hydroxyl end of the primer, and the growth extends across the target DNA. At the end of the first PCR cycle, the initial dsDNA molecule becomes two.

At the completion of this cycle, the reaction mixture is heated again and the 2 existing molecules of DNA will be denatured and the process will repeat again and again.

The DNA polymerase is heat-stable (for example, Taq Taq polymerasepolymerase) from a bacterium that normally lives at high temperatures (a thermophilic bacterium) like Thermus aquaticus, so that it will not be denatured and, therefore, does not have to be added at each successive cycle. Usually 20–30 cycles are run during this process, amplifying the DNA by a million-fold to a billion-fold. The increase in the amount of DNA is exponential with each cycle, hence called “polymerase chain reaction”.

Applications of PCR 1. PCR allows the synthesis of mutant DNA in sufficient

quantities for a sequencing protocol without laboriously cloning the altered DNA.

2. Detection of low-abundance nucleic acid sequences: For example, viruses that have a long latency period, such as human immunodeficiency virus (HIV), are difficult to detect at the early stage of infection using conventional methods. PCR offers a rapid and sensitive method for detecting viral DNA sequences even when only a small proportion of cells is harboring the virus.

3. Forensic analysis of DNA samples: Every individual in this world has what’s called as “DNA

fingerprint” that never repeats in any other person!!! When PCR is used, DNA isolated from only a single human hair, or a tiny spot of blood is quite sufficient to determine the subject to whom such specimens belong. The DNA markers analyzed are most commonly short short tandem repeat polymorphismtandem repeat polymorphism. Verification of paternity uses the same techniques.

4. Prenatal diagnosis and carrier detection of cystic fibrosis:

Cystic fibrosis is an autosomal recessive genetic disease resulting from mutations in the cystic fibrosis cystic fibrosis transmembrane conductance regulatortransmembrane conductance regulator (CFTR)(CFTR) gene. The most common mutation here is a three-base deletion that results in the loss of a phenylalanine residue from the CFTR protein.

Clinically, the patient has thick mucus in his lungs and respiratory passages that is difficult to cough up leading to severe chest infections (mucus is a good medium). The pancreatic digestive enzymes can not pass easily to the intestine (blockage of pancreatic ducts by thick secretions) resulting in defective digestion of food stuff mainly lipids producing malabsorption and steatorrhoea with weight loss.

Slow chronic damage of pancreas leading to diabetes mellitus. Moreover; patients with CF may develop nasal polyps, liver diseases, male and female infertility and osteoporosis.

Diagnosis of cystic fibrosis

Screening

If you have a family history of CF or your partner has CF, you can be tested to see if you carry the CF gene before you start a family.

Testing during pregnancy

If you and your partner are both carriers or if you already have a child with CF, tests can be done early in pregnancy to see if your baby is affected. Tests include the following.1. Amniocentesis - in this test a small sample of the amniotic fluid that surrounds your baby is taken and tested in a laboratory. 2. Chorionic villus sampling - in this test a sample of tissue (biopsy) is taken from the placenta.

Sweat tests

This involves a small amount of sweat being collected from baby's skin and tested for its salt content. People with CF have a large amount of salt in their sweat, so measuring it can help determine whether or not the baby has CF.

If the sweat test indicates that the baby has CF, he or she will be referred to a specialist CF team to discuss available treatments.

As the mutant gene has a shorter allele than normal , it is possible to distinguish them from each other by the size of PCR products obtained by amplifying that portion of DNA.

Treatment of cystic fibrosis

There is currently no cure for CF. There is a lot of research under way to try to find a cure for it through gene therapy or drug therapy to target the CFTR gene. Many of these treatments are undergoing clinical trials.

Current treatments aim to:1. Treat chest infections and prevent further damage to the

lungs. 2. Improve nutrition by providing supplements containing

enzymes to help digestion.

Analysis of Gene Expression

One of the tools of biotechnology is not only to study a gene structure, but also to analyze the products of its expression; mRNA and proteins.

A. Determination of mRNA levels: Messenger RNA levels can be determined by the

hybridization of labeled probes to either mRNA itself or to cDNA produced from mRNA. Techniques used include:

Northern blots: Northern blots are very similar to Southern blots except that the original sample contains a mixture of mRNA molecules that are separated by electrophoresis, then blotted to a membrane and hybridized to a radioactive probe.

Microarrays: It depends on analyzing a sample for the presence of gene mutations (genotyping) or determining the patterns of mRNA production (gene expression) using thousands of immobilized DNA sequences organized in an area no larger than a microscope slide.

For expression analysis, the population of mRNA molecules are first converted to cDNA (by reverse transcriptase) and labeled with a fluorescent tag. This mixture is then exposed to a gene chip, (a glass slide or membrane a glass slide or membrane containing thousands of tiny spots of DNA, each containing thousands of tiny spots of DNA, each corresponding to a different genecorresponding to a different gene).

The amount of fluorescence bound to each spot is a reflection of the amount of that particular mRNA in the sample. DNA microarrays are often used to determine the differing patterns of gene expression in two different types of cell—for example, normal and cancer cells.

B. Analysis of proteins

The types and amounts of proteins in a cell are not always directly proportional to the amounts of mRNA present because some mRNA molecules are translated more efficiently than others, and because some proteins undergo posttranslational modifications by adding sugars or lipids, or both. Thus, the human genome although contains 20,00 to 30,000 genes, but a typical cell produces hundreds of thousands of distinct proteins.

When investigating one, or a limited number of gene products, it is convenient to use labeled antibodies to detect and quantify specific proteins. The present techniques available for this purpose include:

1. Enzyme-linked immunosorbent assays (ELISA):Enzyme-linked immunosorbent assays (ELISA): The antigen (protein) is bound to the wells of the plastic dish. The probe used consists of an antibody specific for the particular protein to be measured. The antibody is covalently bound to an enzyme, which will produce a colored product when exposed to its substrate. The amount of color produced can be used to determine the amount of protein (or antibody) in the sample to be tested.

2. Western blots: Western blots (also called immunoblots) are similar to Southern blots, except that protein molecules in the sample are separated by electrophoresis and blotted (transferred) to a membrane. An antibody bound probe is used producing a band at the location of its antigen.

Proteomics It involves the study of all proteins expressed by

a genome, including their relative abundance, distribution, posttranslational modifications, functions, and interactions with other macromolecules. While a genome remains unchanged, the amounts and types of proteins in any particular cell change dramatically as genes are turned on and off. Proteomics offers the potential of identifying new disease markers and drug targets