Polymerase Chain Reaction

What is PCR (polymerase chain reaction)?

The polymerase chain reaction (PCR) is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNAacross several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence

 PCR (polymerase chain reaction) is a method to analyze a short sequence of DNA (or RNA) even in samples containing only minute quantities of DNA or RNA. PCR is used to reproduce (amplify) selected sections of DNA or RNA.

What molecules take part?

 A basic PCR set up requires several components and reagents. These components include: DNA template that contains the DNA region (target) to be amplified.

 Two primers . short single-stranded DNA sequences that are synthesized to correspond to the beginning and ending of the DNA stretch to be copied Taq polymerase or another DNA polymerase with a temperature optimum at around 70 C. This enzyme called polymerase moves along the segment of DNA, reading its code and assembling a copy.

 Deoxynucleoside triphosphates (dNTPs; nucleotides containing triphosphate groups), the building-blocks from which the DNA polymerase synthesizes a new DNA strand. Buffer solution, providing a suitable chemical environment for optimum activity and stability of the DNA polymerase.

How was PCR (polymerase chain reaction) discovered?

PCR was invented by Kary Mullis. At the time he thought up PCR in 1983, Mullis was working in Emeryville, California for Cetus, one of the first biotechnology companies. There, he was charged with making short chains of DNA for other scientists. Mullis has written that he conceived of PCR while cruising along the Pacific Coast Highway 128 one night on his motorcycle.

 He was playing in his mind with a new way of analyzing changes (mutations) in DNA when he realized that he had instead invented a method of amplifying any DNA region He shared the Nobel Prize in chemistry with Michael Smith in 1993.

 As Mullis has written in the Scientific American: "Beginning with a single molecule of the genetic material DNA, the PCR can generate 100 billion similar molecules in an afternoon. The reaction is easy to execute. It requires no more than a test tube, a few simple reagents, and a source of heat."

What is the purpose of doing a PCR (polymerase chain reaction)?

To do PCR, the original DNA that one wishes to copy need not be pure or abundant. It can be pure but it also can be a minute part of a mixture of materials. So, PCR has found widespread and innumerable uses -- to diagnose genetic diseases, do DNA fingerprinting, find bacteria and viruses, study humanevolution, clone the DNA of an Egyptian mummy, establish paternity or biological relationships, etc..

How is PCR (polymerase chain reaction) done?

Three major steps

30-40 cycles Automated Cycler

Three major steps

1. Denaturation

2. Annealing

3. Extension

Denaturation

At 94 C (201.2 F), the double-stranded DNA melts and opens into two pieces of singlestranded DNA for 20-30sec.

Annealing
At medium temperatures, around 54 C (129.2 F), the primers pair up (anneal) with the single-stranded "template" (The template is the sequence of DNA to be copied.) On the small length of double-stranded DNA (the joined primer and template), the polymerase attaches and starts copying the template.

Extension
The temperature at this step depends on the DNA polymerase used; Taq polymerase has its optimum activity temperature at 75 80 C, and commonly a temperature of 72 C is used with this enzyme. and DNA building blocks complementary to the template are coupled to the primer, making a double stranded DNA molecule.

 With one cycle, a single segment of doublestranded DNA template is amplified into two separate pieces of double-stranded DNA. These two pieces are then available for amplification in the next cycle. As the cycles are repeated, more and more copies are generated and the number of copies of the template is increased exponentially.

Confirmation of formation of DNA

To check whether the PCR generated the anticipated DNA agarose gel electrophoresis is employed for size separation of the PCR products.

PCR stages
The PCR process can be divided into three stages: Exponential amplification: At every cycle, the amount of product is doubled (assuming 100% reaction efficiency). The reaction is very sensitive: only minute quantities of DNA need to be present. Leveling off stage: The reaction slows as the DNA polymerase loses activity and as consumption of reagents such as dNTPs and primers causes them to become limiting.

 Plateau: No more product accumulates due to exhaustion of reagents and enzyme.

PCR optimization

Various techniques for PCR optimization have been developed by molecular biologists to improve PCR performance and minimize failure.

Contamination and PCR

The PCR method is extremely sensitive, requiring only a few DNA molecules in a single reaction for amplification across several orders of magnitude. Therefore, adequate measures to avoid contamination from any DNA present in the lab environment (bacteria, viruses, or human sources) are required.

Division of lab in separate areas

Products from previous PCR amplifications are a common source of contamination, many molecular biology labs have implemented procedures that involve dividing the lab into separate areas. pre-PCR reagents post-PCR processing

SOPs
For the setup of PCR reactions, many standard operating procedures have been developed: which involve 1. pipettes with filter tips 2. wearing fresh laboratory gloves 3. Use of laminar flow cabinet with UV lamp as a work station.(in some cases)

Hairpins
Secondary structures in the DNA can result in folding or knotting of DNA template or primers, leading to decreased product yield or failure of the reaction. Hairpins, which consist of internal folds caused by base-pairing between nucleotides in inverted repeats within single-stranded DNA, are common secondary structures and may result in failed PCR.

Magnesium concentration
Magnesium is required as a co-factor for thermostable DNA polymerase. Taq polymerase is a magnesium-dependent enzyme and determining the optimum concentration to use is critical to the success of the PCR reaction. Some of the components of the reaction mixture such as dNTPs and the presence of chelating agents (EDTA) or proteins can reduce the amount of free magnesium present thus reducing the activity of the enzyme.

Size and other limitations

PCR works readily with a DNA template of up to two to three thousand base pairs in length. However, above this size, product yields often decrease, as with increasing length stochastic effects such as premature termination by the polymerase begin to affect the efficiency of the PCR. It is possible to amplify larger pieces of up to 50,000 base pairs with a slower heating cycle and special polymerases. These are polymerases fused to a processivity-enhancing DNA-binding protein, enhancing adherence of the polymerase to the DNA.

 polymerases TopoTaq include enhanced thermostability, specificity and resistance to contaminants and inhibitors.

Non-specific priming
Non-specific binding of primers frequently occurs and can be due to repeat sequences in the DNA template non-specific binding between primer and template and incomplete primer binding Non-specific binding is also often increased when degenerate primers are used in the PCR.

 Manipulation of annealing temperature and magnesium ion (which stabilise DNA and RNA interactions) concentrations can increase specificity. Nonspecific priming during reaction preparation at lower temperatures can be prevented by using "hot-start" polymerase enzymes whose active site is blocked by an antibody ,once the reaction is heated to 95C during the denaturation step of the first cycle.

 Other methods to increase specificity include

1. Nested PCR 2. Touchdown PCR

Deoxynucleotides
Deoxynucleotides (dNTPs) may bind Mg2+ ions and thus affect the concentration of free magnesium ions in the reaction. In addition, excessive amounts of dNTPs can increase the error rate of DNA polymerase and even inhibit the reaction. An imbalance in the proportion of the four dNTPs can result in misincorporation into the newly formed DNA strand and contribute to a decrease in the fidelity(accuracy) of DNA polymerase.

What is RT PCR?
RT-PCR (Reverse transcriptase-polymerase chain reaction) is a highly sensitive technique for the detection and quantitation of mRNA (messenger RNA).

The technique consists of two parts: The synthesis of cDNA (complementary DNA) from RNA by reverse transcription (RT) The amplification of a specific cDNA by the polymerase chain reaction (PCR).

Uses of RT
RT-PCR has been used to measure viral load with HIV may also be used with other RNA viruses such as measles and mumps.

Application of PCR

The Polymerase Chain Reaction (PCR) has foundwidespread application in many areas of genetic analysis. This is a list of some of these applications:

1. 2. 3. 4. 5.

Medical applications Infectious disease applications Forensic applications Research applications Others

1. Medical applications
The first application of PCR was for genetic testing, where a sample of DNA is analyzed for the presence of genetic disease mutations. Prospective parents can be tested for being genetic carriers, or their children might be tested for actually being affected by a disease.

 DNA samples for Prenatal testing can be obtained by I. amniocentesis, (also referred to as amniotic fluid test or AFT) is a medical procedure used in prenatal diagnosis of chromosomal abnormalities and fetal infections, in which a small amount of amniotic fluid, which contains fetal tissues, is sampled from the amnion or amniotic sac surrounding a developing fetus, and the fetal DNA is examined for genetic abnormalities. Using this process the sex of the fetus can also be determined and hence this procedure has legal restrictions in some gender-biased countries.)

I. chorionic villus sampling II. even by the analysis of rare fetal cells circulating in the mother's bloodstream. PCR analysis is also essential to Preimplantation genetic diagnosis, where individual cells of a developing embryo are tested for mutations.

 PCR can also be used as part of a sensitive test for tissue typing, vital to organ transplantation.

Many forms of cancer involve alterations to oncogenes. By using PCR-based tests to study these mutations, therapy regimens can sometimes be individually customized to a patient.

2. Infectious disease applications

I. Infections can be detected earlier II. donated blood can be screened directly for the virus III. newborns can be immediately tested for infection IV. the effects of antiviral treatments can be quantified.

V .Some disease organisms, such as that for Tuberculosis, are difficult to sample from patients and slow to be grown in the laboratory. PCR-based tests have allowed detection of small numbers of disease organisms (both live or dead), in convenient samples

 VI. The spread of a disease organism through populations of domestic or wild animals can be monitored by PCR testing. In many cases, the appearance of new virulent sub-types can be detected and monitored.

3. Forensic applications

i. Used for genetic (or DNA) fingerprinting. helpful in crime scene

4. Research Applications
i. PCR allows rapid production of short pieces of DNA, even when nothing more than the sequence of the two primers is known. This ability of PCR augments many methods, such as generatinghybridization probes for Southern or northern blot hybridization
(A Southern blot is a method routinely used in molecular biology for detection of a specific DNA sequence in DNA samples.) The northern blot is a technique used in molecular biology research to study gene expression by detection of RNA (or isolated mRNA) in a sample)

ii. The task of DNA sequencing can also be assisted by PCR. Known segments of DNA can easily be produced from a patient with a genetic disease mutation. iii. PCR has numerous applications to the more traditional process of DNA cloning. It can extract segments for insertion into a vector from a larger genome, which may be only available in small quantities. Using a single set of 'vector primers', it can also analyze or extract fragments that have already been inserted into vectors. Some alterations to the PCR protocol can generate mutations (general or site-directed) of an inserted fragment.

iv. Sequence-tagged sites is a process where PCR is used as an indicator that a particular segment of a genome is present in a particular clone.

v.

An exciting application of PCR is the phylogenic analysis of DNA from ancient sources. ( is the study of evolutionary relation among groups of organisms)

Vi . A common application of PCR is the study of patterns of gene expression. Tissues (or even individual cells) can be analyzed at different stages to see which genes have become active, or which have been switched off.

5. Others
i. In the field of evolutionary biology, PCR has been used to establish relationships among species.

ii.

In anthropology, it has been used to understand ancient human migration patterns.

iii. In archaeology, it has been used to help identify ancient human remains.

iv. Paleontologists have used PCR to amplify DNA from extinct insects preserved in amber for 20 million years. v. The genes responsible for a variety of human diseases have been identified using PCR. For example, a PCR technique called multiplex PCR identifies a mutation in a gene in boys suffering from Duchenne muscular dystrophy. vi. PCR can also be used to search for DNA from foreign organisms such as viruses or bacteria

Variations on the basic PCR technique

1. Assembly PCR or Polymerase Cycling Assembly (PCA) 2. Asymmetric PCR 3. Helicase-dependent amplification 4. Hot start PCR 5. Intersequence-specific PCR 6. Inverse PCR

7. Ligation-mediated PCR 8. Multiplex-PCR 9. Nested PCR 10.Overlap-extension PCR 11.Quantitative PCR 12.Reverse Transcription PCR (RT-PCR) 13.Solid Phase PCR

14.Thermal asymmetric interlaced PCR (TAILPCR) 15.Touchdown PCR (Step-down PCR) 16.PAN-AC 17.Universal Fast Walking 18.In silico PCR

Thanks