Question

One of the powerful tools in biomolecule engineering is Polymerase Chain Reactions (PCR). What is PCR and describe its importance of this technique. Using a proper example, discuss one (1) usage of PCR technology in the crime/forensic or detection of diseases.

Answer 1

Polymerase Chain Reactions (PCR) and its importance: PCR is a simple but very useful technique in molecular biology and an important tool for many applications. PCR was invented in 1983 by the American biochemist Kary Mullis at Cetus Corporation. It is a technique used to amplify a segment of DNA of interest or produce lots and lots of copies. PCR can be used to amplify a sample of DNA when there isn’t enough to analyze (e.g. a sample of DNA from a crime scene, archeological samples), as a method of identifying a gene of interest, or to test for disease In other words, PCR enables you to produce millions of copies of a specific DNA sequence from an initially small sample – sometimes even a single copy. It is a crucial process for a range of genetic technologies and, in fact, has enabled the development of a suite of new technologies.

Importance of PCR: PCR have a wide range of specialized applications and are used by scientists in all fields of biology. E.g. in Forensic science, in phylogenetics, in medicine, in many environmental issues, genomics etc. Few example are listed below.

1. PCR has become an important tool for medical diagnosis. PCR can detect and identify bacteria and viruses that cause infections such as tuberculosis, viral meningitis, viral hepatitis, HIV, etc.

2. PCR is also used in genetic testing, to determine whether patients carry a genetic mutation that could be passed on to their children (e.g. the mutation that causes cystic fibrosis) or to determine disease risk in patients themselves (e.g. a mutation in the gene BRCA1 predisposes a woman to breast or ovarian cancer).

3. PCR is used in genome sequencing, including the Human Genome Project. Using random primers (not a specific sequence), the entire genome of an organism can be amplified in pieces.

4. Scientists can gather information about evolutionary relationships using PCR on ancient samples. Genes from various related organisms are amplified, sequenced and then analyzed for similarities/differences.

They way PCR works: PCR mimics what happens in cells when DNA is copied (replicated) prior to cell division, but it is carried out in controlled conditions in a laboratory. The machine that is used is simply called a PCR machine or a thermocycler. Test tubes containing the DNA mixture of interest are put into the machine, and the machine changes the temperature to suit each step of the process.

The method uses specifically designed primers that are complementary to the sequence to be amplified. The primers provide a starting point for the extension of the DNA by a DNA polymerase (usually Taq or Pfu polymerase). Amplification is carried out in cycles. First, the DNA sample is heated up to separate the double strands. The sample is cooled slowly, allowing the primers to bind. Then, the sample is incubated at 72°C so that the DNA Polymerase can extend the primers, creating a long complementary strand of DNA. In this way, one double strand of DNA becomes 2, 2 become 4, 4 become 8 and so on.

Standard ingredients in the mixture are:

· the DNA segment of interest

· specific primers: two DNA primers that are complementary to the 3' ends of each of the sense and anti-sense strands of the DNA target.

· heat-resistant DNA polymerase enzyme

· the four different types of DNA nucleotides (dNTPs)

· the salts needed to create a suitable environment for the enzyme to act (suitable buffer).

PCR process: 3 main steps of PCR (see figure)

Step 1: Denaturation (96 °C)

Initialization step needed required for heat activation of DNA polymerases by hot-start. It consists of heating the reaction chamber to a temperature of 94–96 °C, or 98 °C.

As in DNA replication, the two strands in the DNA double helix need to be separated. The separation happens by raising the temperature of the mixture, causing the hydrogen bonds between the complementary DNA strands to break. This process is called denaturation. It consists of heating the reaction chamber to 94–98 °C for 20–30 seconds.

Step 2: Annealing (mostly 55-65 °C)

Primers bind to the target DNA sequences and initiate polymerization. This can only occur once the temperature of the solution has been lowered. One primer binds to each strand. In this step, the reaction temperature is lowered to 50–65 °C for 20–40 seconds, allowing annealing of the primers to each of the single-stranded DNA templates. It is critical to determine a proper temperature for the annealing step because efficiency and specificity are strongly affected by the annealing temperature. This temperature must be low enough to allow for hybridization of the primer to the strand, but high enough for the hybridization to be specific, i.e., the primer should bind only to a perfectly complementary part of the strand, and nowhere else. If the temperature is too low, the primer may bind imperfectly. If it is too high, the primer may not bind at all. A typical annealing temperature is about 3–5 °C below the Tm of the primers used.

Step 3: Extension (72 °C)

New strands of DNA are made using the original strands as templates. A DNA polymerase enzyme joins free DNA nucleotides together. This enzyme is often Taq polymerase, an enzyme originally isolated from thermophilic bacteria called Thermus aquaticus.

The result of one cycle of PCR is two double-stranded sequences of target DNA, each containing one newly made strand and one original strand.

The processes of denaturation, annealing and elongation constitute a single cycle. The cycle is repeated many times (usually 25–35) as most processes using PCR need large quantities of DNA. It only takes 2–3 hours to get a billion or so copies. The formula used to calculate the number of DNA copies formed after a given number of cycles is 2ⁿ, where n is the number of cycles. Thus, a reaction set for 20 cycles results in 2,097,152 copies of the original double-stranded DNA target region.

Final elongation: This step is performed at a temperature of 70–74 °C for 5–10 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully elongated.

Forensic science: PCR is very important for the identification of criminals and the collection of organic crime scene evidence such as blood, hair, pollen, semen and soil. DNA fingerprints (also called DNA profiles), identification of familial relationships, genomic DNA isolation and other molecular diagnostics and biochemical analyses can be undertaken forensically through the use of PCR. Forensics experts can only compare crime scene DNA with a databank of DNA samples from known criminals.

For example, one PCR based technique is Short tandem repeat (STR) technology; it is a forensic analysis that evaluates specific regions (loci) that are found on nuclear DNA. The variable (polymorphic) nature of the STR regions that are analyzed for forensic testing intensifies the discrimination between one DNA profile and another. In order to match, for example, crime scene evidence to a suspect, a lab would determine the allele profile of the 13 core STRs for both the evidence sample and the suspect's sample. If the STR alleles do not match between the two samples, the individual would be excluded as the source of the crime scene evidence. However, if the two samples have matching alleles a statistical calculation would be made to determine probability of presence of suspect on the crime site.