Optimizing a Conventional Polymerase Chain Reaction (PCR) and Primer Design

Transcription

1 Optimizing a Conventional Polymerase Chain Reaction (PCR) and Primer Design The Polymerase Chain Reaction (PCR) is a powerful technique used for the amplification of a specific segment of a nucleic acid usually DNA (in conventional PCR) or complementary DNA "cdna" (which is produced from RNA by reverse transcriptase) in RT- PCR. The aim behind conducting PCR is to get a large number (10 9 ) of DNA copies so that they can be analyzed more easily later on by the downstream procedures like sequencing or digestion by restriction endonucleases. Because of its great sensitivity, PCR has found popularity in a wide range of medical, biological and forensic applications as in gene cloning, DNA sequencing and when PCR is used to connect blood, saliva, hair or other tissues left at the place of a crime to a suspect or victim. Simply, PCR is DNA synthesis in a test tube. To perform PCR amplification, we need to get all the following components in the reaction tube: 1. Template DNA. The template carries the DNA segment that we want to amplify. This DNA is the "Target DNA sequence". 2. Primers. A primer is a short (oligonucleotide), single-stranded DNA that anneals (attaches) to a specific DNA sequence in a complementary way. A pair of primers will bind to either side of the target DNA segment providing initiation sites for DNA synthesis. The two primers are designated by the terms forward and reverse. Sometimes we call them as "Sense" and "Antisense". 3. DNA Polymerase. This is the enzyme used to synthesize new strands of DNA. Its job is to add nucleotides onto the 3ˋend of an annealed primer. The added bases are complementary to those in the template strand. Taq polymerase is the most widely used with its modifications (e.g. HotStar Taq Polymerase). This enzyme is either bacterial in origin or recombinant. Page 1

2 4. Magnesium. DNA polymerase requires magnesium for its activity. Magnesium is usually supplied in the form of magnesium chloride (MgCl2). 5. dntps. They stand for "deoxyribonucleoside triphosphate". These are the four bases used by DNA polymerase to extend an annealed primer. They are datp, dgtp, dctp, dttp. 6. Buffer. A buffer is a solution that resists change in ph. It is used to offer a constant ph at which the activity of DNA polymerase will be maximal. When developing a protocol for PCR amplification of a new target, it is very important to optimize all parameters including reagent concentrations, cycling temperatures, and cycle number. The thermal cycles are conducted inside a thermal cycler which is the PCR machine, (Fig.1). Figure 1. A photograph of a thermal cycler (conventional PCR machine) Page 2

3 Each thermal cycle includes three phases; denaturation, annealing and extension and they are preceded by an "initial denaturation step" and followed by a step of "final extension", Fig.2. Figure 2. An example of a thermal cycling programme for a PCR. During optimization, varying the magnesium or primer concentration usually profound effects on the quality of the reaction and for successful PCR, primers must be added in molar excess over the amount of target DNA. This ensures that, following the denaturation step primer/template annealing is favored over template/template reannealing. An optimized PCR amplification will produce a single bright band on a gel (following gel electrophoresis) with the exact desired size. On the contrary, a reaction for which conditions have not been optimized will produce either no band or multiple non- specific bands, Fig.(3). Figure 3. A gel of successful (most left) and failed (central) PCRs *The most right is the DNA size marker Page 3

4 How to design primers for PCR to amplify a target DNA sequence? When we have a target sequence that we want to amplify, a pair of primers have to be designed carefully to anneal efficiently and specifically either using a commercial software (e.g. Primer 3.0) or manually. The primers have to fulfill the following criteria: 1. The primers have to be designed to amplify the target DNA sequence in a particular gene of a particular species (i.e species, gene and sequence specific). The size of the amplicon they would produce has to be proper depending on where the primers attach. 2. The ideal primer length is bases. 3. The GC ra o has to be 40-60%. 4. The primers should end with one or two C or G residue, because T and A residues can bind more easily to DNA in a non-specific way. 5. Both forward and reverse primers should have the same melting temperature (Tm). However, if a difference is there, it should not exceed 3 C for real- me PCR or 5 C for conventional PCR. 6. They (forward and reverse primers) have to be sequence specific i.e. should be able to amplify one sequence in the whole genome only. Specificity of primers is checked by referring to softwares like NCBI (National Center for Bio-Informatics) Blast. 7. They should exhibit the lowest "self complementarity" i.e least possibilities of forming secondary structures as primer dimmers or hairpins mainly at their 3ˋ end (because primers are extended on this end). Softwares like GenRunner can help to analyze the oligonucleotides (primers and probes) for self-complementarity beside other parameters. Page 4

5 8. Primers should be tested for their ability to produce the desired amplicon on paper first before being ordered and added in the experimental reaction tube using softwares like InSilico PCR. This is what is termed as dry PCR. 9. Depending on the use of the primers, the method of their purification has to be chosen carefully. DHPLC is used for purification of primers to be used for sequencing (probes). For less sensitive post-pcr applications like enzyme digestion, desalting of primers may be fair enough. Estimation of Primer Melting Temperature (Tm) For primers containing less than 25 nucleo des, the approximate melting temperature (Tm) can be calculated using the following equation: Tm = 4 (G + C) + 2 (A + T), where G, C, A, T are the respective nucleotides in the primer. If the primer contains more than 25 nucleo des, specialized computer programs e.g, REviewer are recommended. In general, a primer's annealing temperature (Ta) ranges between "Tm - 5C " to "Tm + 5C ". This is the range that we have to go through while optimizing a thermal cycling program. In summary, to optimize a conventional PCR, one has to keep in mind the following points: Page 5 Optimally design highly specific primers (will be practiced) High Tm of primers (and thus high Ta) would improve their specificity, however if two high, there will be less PCR product and if two low, there might be so many non-specific bands. When the amount of the PCR product is lower than expected (faint band on gel), the best solution is to either to increase the

6 amount of MgCl2, increase the PCR cycles number or lower the annealing temperature. Non-specific bands are formed by either poor- quality primers (non-specific) or because of bad cycling program. Increasing the annealing temperature can usually solve the problem if the primers are OK. Page 6