Polymerase chain reaction (PCR)

PCR

Definition :

PCR is a laboratory method used to make many copies of a specific DNA sequence. This method serves many purposes including disease diagnosis, detection of difficult-to-isolate pathogens (any disease-producing microorganism) , mutation analysis, genetic testing, DNA sequencing, and analyzing evolutionary relationships.

Polymerase chain reaction (PCR) is a molecular biology technique for enzymatically replicating DNA without using a living organism, such as E. coli or yeast. Like amplification using living organisms, the technique allows a small amount of DNA to be amplified exponentially. As PCR is an in vitro technique, it can be performed without restrictions on the form of DNA and it can be extensively modified to perform a wide array of genetic manipulations.ِ

Inventor :

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 1 one night in his car. 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. Mullis has said that before his trip was over, he was already savoring the prospects of a Nobel Prize. He shared the Nobel Prize in Chemistry with Michael Smith in 1993.

Patent wars :

The PCR technique was patented by Cetus Corporation, where Mullis worked when he invented the technique in 1983. The Taq polymerase enzyme is also covered by patents. There have been several high-profile lawsuits related to the technique, including an unsuccessful lawsuit brought by DuPont. The pharmaceutical company Hoffmann-La Roche purchased the rights to the patents in 1992 and currently holds those that are still protected.

What does PCR need in work ?

PCR is used to amplify specific regions of a DNA strand. This can be a single gene, just a part of a gene, or non-coding sequence. PCR typically amplifies only short DNA fragments, usually up to 10 kilo base pairs (kb). Certain methods can copy fragments up to 47 kb in size, which is still much less than the chromosomal DNA of a eukaryotic cell - for example, a human cell contains about three billion base pairs.

PCR, as currently practiced, requires several basic components. These components are:

• DNA template, which contains the region of the DNA fragment to be amplified
• Two primers, which determine the beginning and end of the region to be amplified (see following section on primers)
• Taq polymerase (or another durable polymerase), a DNA polymerase, which copies the region to be amplified
• Deoxynucleotide triphosphates, (dNTPs) from which the DNA polymerase builds the new DNA
• Buffer, which provides a suitable chemical environment for the DNA Polymerase

The PCR process is carried out in a thermal cycler. This is a machine that heats and cools the reaction tubes within it to the precise temperature required for each step of the reaction. To prevent evaporation of the reaction mixture (typically volumes between 15-100µl per tube), a heated lid is placed on top of the reaction tubes, or a layer of oil is put on the surface of the reaction mixture. These machines cost more than $2,500 USD, as of 2004.

What are primers ?

The DNA fragment to be amplified is determined by selecting primers. Primers are short, artificial DNA strands — often not more than 50 and usually only 18 to 25 base pairs long — that are complementary to the beginning or the end of the DNA fragment to be amplified. They anneal by adhering to the DNA template at these starting and ending points, where the DNA polymerase binds and begins the synthesis of the new DNA strand.

The choice of the length of the primers and their melting temperature (T_m) depends on a number of considerations. The melting temperature of a primer -- not to be confused with the melting temperature of the template DNA -- is defined as the temperature at which half of the primer binding sites are occupied. Primers that are too short would anneal at several positions on a long DNA template, which would result in non-specific copies. On the other hand, the length of a primer is limited by the maximum temperature allowed to be applied in order to melt it, as melting temperature increases with the length of the primer. Melting temperatures that are too high, i.e., above 80°C, can cause problems since the DNA polymerase is less active at such temperatures. The optimum length of a primer is generally from 15 to 40 nucleotides with a melting temperature between 55°C and 65°C.

Procedure :

The PCR process usually consists of a series of twenty to thirty-five cycles. Each cycle consists of three steps (Fig. 2).

1. The double-stranded DNA has to be heated to 94-96°C (or 98°C if extremely thermostable polymerases are used) in order to separate the strands. This step is called denaturing; it breaks apart the hydrogen bonds that connect the two DNA strands. Prior to the first cycle, the DNA is often denatured for an extended time to ensure that both the template DNA and the primers have completely separated and are now single-strand only. Time: usually 1-2 minutes, but up to 5 minutes. Also certain polymerases are activated at this step (see hot-start PCR).
2. After separating the DNA strands, the temperature is lowered so the primers can attach themselves to the single DNA strands. This step is called annealing. The temperature of this stage depends on the primers and is usually 5°C below their melting temperature (45-60°C). A wrong temperature during the annealing step can result in primers not binding to the template DNA at all, or binding at random. Time: 1-2 minutes.
3. Finally, the DNA polymerase has to copy the DNA strands. It starts at the annealed primer and works its way along the DNA strand. This step is called elongation. The elongation temperature depends on the DNA polymerase. Taq polymerase elongates optimally at a temperature of 72 Celsius. The time for this step depends both on the DNA polymerase itself and on the length of the DNA fragment to be amplified. As a rule-of-thumb, this step takes 1 minute per thousand base pairs. A final elongation step is frequently used after the last cycle to ensure that any remaining single stranded DNA is completely copied. This differs from all other elongation steps, only in that it is longer, typically 10-15 minutes. This last step is highly recommendable if the PCR product is to be ligated into a T vector using TA-cloning.

Figure 2: Schematic drawing of the PCR cycle. (1) Denaturing at 94-96°C. (2) Annealing at (eg) 68°C. (3) Elongation at 72°C (P=Polymerase). (4) The first cycle is complete. The two resulting DNA strands make up the template DNA for the next cycle, thus doubling the amount of DNA duplicated for each new cycle (a total of three cycles is shown above).

Steps of PCR process :

The PCR process consists of the following steps:

1. Initialization. The mixture is heated at 96°C for 5 minutes to ensure that the DNA strands as well as the primers have melted. The DNA Polymerase can be present at initialization, or it can be added after this step.
2. Melting, where it is heated at 96°C for 30 seconds. For each cycle, this is usually enough time for the DNA to denature.
3. Annealing by heating at 68°C for 30 seconds:The primers are jiggling around, caused by the Brownian motion. Short bondings are constantly formed and broken between the single stranded primer and the single stranded template. The more stable bonds last a little bit longer (primers that fit exactly) and on that little piece of double stranded DNA (template and primer), the polymerase can attach and starts copying the template. Once there are a few bases built in, the Tm of the double-stranded region between the template and the primer is greater than the annealing or extension temperature.
4. Elongation by heating 72°C for 45 seconds:This is the ideal working temperature for the polymerase. The primers, having been extended for a few bases, already have a stronger hydrogen bond to the template than the forces breaking these attractions. Primers that are on positions with no exact match, melt away from the template (because of the higher temperature) and are not extended.

The bases (complementary to the template) are coupled to the primer on the 3' side (the polymerase adds dNTP's from 5' to 3', reading the template from 3' to 5' side, bases are added complementary to the template)

1. Steps 2-4 are repeated 25 times, but with good primers and fresh polymerase, 15 to 20 cycles is sufficient.
2. Mixture is held at 7°C. This is useful if one starts the PCR in the evening just before leaving the lab, so it can run overnight. The DNA will not be damaged at 7°C after just one night.

The PCR product can be identified by its size using agarose gel electrophoresis. Agarose gel electrophoresis is a procedure that consists of injecting DNA into agarose gel and then applying an electric current to the gel. As a result, the smaller DNA strands move faster than the larger strands through the gel toward the positive current. The size of the PCR product can be determined by comparing it with a DNA ladder, which contains DNA fragments of known size.

PCR optimization :

Since PCR is very sensitive, adequate measures to avoid contamination from other DNA present in the lab environment (bacteria, viruses, lab staff's skin etc.) should be taken. Thus DNA sample preparation, reaction mixture assemblage and the PCR process, in addition to the subsequent reaction product analysis, should be performed in separate areas. For the preparation of reaction mixture, a laminar flow cabinet with UV lamp is recommended. Fresh gloves should be used for each PCR step as well as displacement pipettes with aerosol filters. The reagents for PCR should be prepared separately and used solely for this purpose. Aliquots should be stored separately from other DNA samples. A control reaction (inner control), omitting template DNA, should always be performed, to confirm the absence of contamination or primer multimer formation .

Difficulties with polymerase chain reaction :

Polymerase chain reaction is not perfect, and errors and mistakes can occur. These are some common errors and problems that may occur.

1- Polymerase errors

Taq polymerase lacks a 3' to 5' exonuclease activity. This makes it impossible for it to check the base it has inserted and remove it if it is incorrect, a process common in higher organisms. This in turn results in a high error rate of approximately 1 in 10,000 bases, which, if an error occurs early, can alter large proportions of the final product.

2-Size limitations

PCR works readily with DNA of lengths two to three thousand base pairs, but above this length the polymerase tends to fall off, and the typical heating cycle does not leave enough time for polymerisation to complete. It is possible to amplify larger pieces of up to 50,000 base pairs with a slower heating cycle and special polymerases. These special polymerases are often polymerases fused to a DNA-binding protein, making them literally "stick" to the DNA longer.

3- Non specific priming

The non specific binding of primers is always a possibility due to sequence duplications, non-specific binding and partial primer binding, leaving the 5' end unattached. This is increased by the use of degenerate sequences or bases in the primer.

Practical modifications to the PCR technique :

• Nested PCR - Nested PCR is intended to reduce the contaminations in products due to the amplification of unexpected primer binding sites.
• Intersequence specific (ISSR) PCR
• Ligation-mediated PCR

• Inverse PCR - Inverse PCR is a method used to allow PCR when only one internal sequence is known.

• RT-PCR - RT-PCR (Reverse Transcription PCR) is the method used to amplify, isolate or identify a known sequence from a cell or tissues RNA library.

• Assembly PCR - Assembly PCR is the completely artificial synthesis of long gene products by performing PCR on a pool of long oligonucleotides with short overlapping segments.

• Asymmetric PCR - Asymmetric PCR is used to preferentially amplify one strand of the original DNA more than the other.

• Quantitative PCR - Q-PCR (Quantitative PCR) is used to rapidly measure the quantity of PCR product (preferably real-time), thus is an indirect method for quantitatively measuring starting amounts of DNA, cDNA or RNA.

• Quantitative real-time PCR is often confusingly known as RT-PCR (Real Time PCR) and RQ-PCR. QRT-PCR or RTQ-PCR are more appropriate contractions. This method uses fluorescent dyes and probes to measure the amount of amplified product in real time.

• Touchdown PCR - Touchdown PCR is a variant of PCR that reduces nonspecific primer annealing by more gradually lowering the annealing temperature between cycles.

• Hot-start PCR is a technique that reduces non-specific priming that occurs during the preparation of the reaction components.

• Colony PCR - Bacterial clones (E.coli) can be screened for the correct ligation products.

• RACE-PCR - Rapid amplification of cDNA ends.

• Multiplex-PCR - The use of multiple, unique primer sets within a single PCR reaction to produce amplicons of varying sizes specific to different DNA sequences.

• Methylation Specific PCR - Methylation Specific PCR (MSP) is used to detect methylation of CpG islands in genomic DNA.

Uses of PCR :

PCR can be used for a broad variety of experiments and analyses. Some examples are discussed below.

1- Genetic fingerprinting

Genetic fingerprinting is a forensic technique used to identify a person by comparing his or her DNA with a given sample.

2- Detection of hereditary diseases

The detection of hereditary diseases in a given genome is a long and difficult process, which can be shortened significantly by using PCR.

Viral diseases, too, can be detected using PCR through amplification of the viral DNA. This analysis is possible right after infection, which can be from several days to several months before actual symptoms occur. Such early diagnoses give physicians a significant lead in treatment.

3- Cloning genes

4- Mutagenesis

Mutagenesis is a way of making changes to the sequence of nucleotides in the DNA. There are situations in which one is interested in mutated (changed) copies of a given DNA strand, for example, when trying to assess the function of a gene or in in-vitro protein evolution (also known as Directed evolution).

5- Analysis of ancient DNA

Using PCR, it becomes possible to analyze DNA that is thousands of years old. PCR techniques have been successfully used on animals, such as a forty-thousand-year-old mammoth, and also on human DNA, in applications ranging from the analysis of Egyptian mummies to the identification of a Russian Tsar.

6- Genotyping of specific mutations

Through the use of allele-specific PCR, one can easily determine which allele of a mutation or polymorphism an individual has.

7- Comparison of gene expression

Researchers have used traditional PCR as a way to estimate changes in the amount of a gene's expression. Ribonucleic acid (RNA) is the molecule into which DNA is transcribed prior to making a protein.

References :

1- www.pcrstation.com

2- www.pcrlinks.com

3- www. en.wikipedia.org

4- www.genome.gov

5- www.dnalc.org

6- www.pcrnewsletter.com

7- www.horizonpress.com

8- www.uq.edu.au