Polymerase Chain Reaction (PCR)
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:It permits the synthesis of millions of copies of a specific nucleotide sequence in a relatively short time (a few hours). 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) The method can be used to amplify DNA sequences from any source—bacterial, viral, plant, or animal.Steps of PCR
1. The first step of PCR is to denature the DNA (or DNA melting), where the DNA to be amplified is heated by rising the temperature to as high as around 92 cє to separate the double-stranded target DNA into single strands.2. Annealing of “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 primers in PCR reactions. The 3′-hydroxyl end of each primer points toward the target sequence. The separated strands are cooled down (like to 60 cє ) and allowed to anneal to the two primers (one primer for each strand).
3. Chain extension: Both DNA polymerase and deoxyribonucleoside triphosphates (dNTPs) (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 i.e melted and the process will repeat again and again. The DNA polymerase is heat-stable (for example, Taq polymerase) 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. Note: The DNA markers analyzed are most commonly short tandem repeat polymorphism. Verification of paternity uses the same techniques.
4. Prenatal diagnosis and carrier detection of genetic diseases (as example: cystic fibrosis):
Cystic fibrosis is an autosomal recessive multisystemic genetic disease resulting from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene (63 bp). ***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 (before marriage) 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 (Prenatal) 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 the 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. Note: The aim is discover the presence of the mutation of the CFTR gene in the fetal cells
As the mutant gene has a shorter allele than normal , it is possible to distinguish them from each other by the size of the PCR products obtained by amplifying that portion of DNA.
Sweat test: 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.
Treatment of cystic fibrosis There is currently no cure for CF. However, 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 (enzyme replacement therapy).
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 (i.e short ssDNA stretches labeled mostly with a fluorescent dye like TaqMan Probes) to either mRNA itself or to cDNA produced from mRNA. Techniques used include:
Real- Time PCR: It counts the number of copies of mRNA molecules per time unit. Over-expressed genes give higher number of copies while under-expressed genes give a low- copy number. So; high- copy number gene low- copy number gene
2. Northern blots: Northern blots are very similar to Southern blots (a method for measuring DNA invented by the scientist “SOUTHERN”) except that the original sample contains a mixture of mRNA molecules that are separated by electrophoresis, then blotted (i.e transferred) to a membrane (like nitrocellulose) and hybridized to a radioactive probe (i.e a stretch of DNA attached to a radioactive isotope). The higher radioactivity measured indicates the larger amount of mRNA in the mixture to start with (i.e. higher gene expression).
3. Microarrays: It’s a technique used for:a. analyzing a sample for the presence of gene mutations (genotyping) b. or determining the patterns of mRNA production (gene expression)
This method relies on using thousands of immobilized DNA sequences (known) organized in an area no larger than a microscope slide known as the “gene chip” which is either of glass or membranous material.
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, (containing thousands of tiny spots of DNA, each corresponding 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.
B. Analysis of proteins The study of gene expression does not only imply mRNA measurement. It has to include also the study of the level of mRNA translation i.e. the efficacy of protein production. 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 20,000 to 30,000 genes, but a typical cell produces hundreds of thousands of distinct proteins.
When investigating one, or a limited number of proteins, 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): 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 when stained then with a special stain.