Maintaining a high annealing temperature in second phase prevents this contamination. This second primer basically amplifies a specific region inside the genomic sequence that was amplified.īut this process has a disadvantage that it causes contamination of sample when transferred to separate tube for second phase of amplification. The principle is that the amplified product of the first primer is subjected to second amplification cycle that applies second set of primer in a separate tube. This type is based upon the principle of double amplification that is accomplished using two different sets of primers. Moreover, each cycle is repeated twice and the number of repeating cycles is controlled. In this type of PCR reaction, the initial or the annealing temperature is kept higher as compared to the standard temperature and this temperature is lowered systematically in each cycle. If a single strand is to be amplified, primers are added both for forward and reverse transcriptions to amplify a specific region within the same template strand.Īs indicated by its name, it can amplify multiple template strands by application of multiple primers but in the same reaction mixture. It also has applications for identification of exonic and the intronic sequences in DNA strand.ĭepending upon the number and the type of primer required in the process, it can be classified as: This type is useful for simultaneous amplification of multiple sequences within the same sample. In this way a small quantity of DNA template can be amplified. In this type both the strands of DNA are amplified using two separate poly dT primers for each of them. Depending upon the type of transcriptase enzyme it can amplify genomic sequences ranging from 1-12 kbs. The enzyme used in this PCR reaction is RNA dependent polymerase obtained from thermus themophillus bacterial species. So it is basically used to amplify and identify the genomic sequence of viruses and retroviruses that become incorporated in the host genome. This type is based upon the principle of reverse transcription of gene that is amplified. Here are some types of PCR that are based upon their type of genetic sequence they amplify and their specificity as well: DNA bases & PCR machine that amplifies the gene number.Taq polymerase enzyme that adds DNA bases to the growing strand of DNA and can withstand the high temperature of reaction mixture required to unwind DNA.Primer sequence to bind to 3’ end of DNA strand from where the replication will start.This cycle repeats itself for about 35 times to get millions of copy of the initial DNA sequence. These three steps constitute one cycle of replication. The basic steps in all the types of polymerase chain reaction are almost the same. Basic steps of Polymerase chain reaction (PCR) The repeated replication of one particular sequence of DNA provides millions of its copies that can be used in microbiological and molecular researches and in field of forensic science also. Most of the PCR reactions can amplify the genetic sequence of 0.1 to 10 kbs and this range can be up to 40 kbs. The basic principle of this technique is that the DNA replicates itself with the help of polymerase enzyme using its bases and the primer sequence. Kary Mullis developed this technique in 1938. Polymerase Chain Reaction (PCR) is a biotechnological technique which amplifies a particular sequence of DNA and produces millions of copies of specific gene sequence.
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