PCR cloning is a method of cloning which dramatically reduces the time and effort put into the cloning reaction. PCR cloning procedure consisting of the four following steps: (1) production of a fragment of the gene using PCR, (2) digestion of genomic DNA, (3) ligation into a plasmid vector, and (4) transformation into bacteria and then bacteria will replicate the plasmid.
The procedure of PCR cloning includes DNA preparation, digestion, ligation and transformation. This video shows the first part- insert PCR. Compared to other Taq-generated PCR inserts, Pfu DNA polymerase-generated PCR fragments will have fewer errors and is more commonly used for molecular cloning.
Digestion is the process of cutting DNA molecules with restriction endonucleases. These special enzymes recognize specific sequences in the DNA molecule (for example GATATC) wherever that sequence occurs in the DNA. In PCR cloning, insert and vector are cutted by the same restriction endonucleases and then join together in the next step, ligation.
DNA ligation joins two linear DNA fragments together with covalent phosphodiester bonds between 3 hydroxyl ends of one nucleotide with the 5 phosphate end of another. In PCR cloning, insert DNA and vector digested by the same restriction enzyme can be ligated by DNA ligase.
The purpose of transformation is to introduce a foreign plasmid into bacteria and the bacteria will replicate the foreign plasmid along with their own DNA. Bacteria which are able to uptake DNA after a heat shock are called "competent".
It can be used after a transformation to screen colonies for the plasmid. Primers designed for the insert sequence should be used when preparing the PCR reaction. Thus, any colonies which give rise to an amplification product are likely to contain the correct DNA sequence.
PCR can be used to make changes to the nucleotide sequence of DNA. This is called PCR mutagenesis. Site-direct mutagenesis is one kind of PCR mutagenesis, which introduces a mutation at a specific location on the DNA strand.
Following PCR, you need to get rid of excess short primers, dNTPs, enzymes, short-failed PCR products and salts. We use a silica-gel-membrane for binding of DNA in high-salt buffer (pH 4.5~5.5) and elution of DNA with low-salt buffer (pH 7.0~9.0) or ddH2O to get clean PCR products for downstream applications.
Polymerase Chain Reaction (PCR) is a technique to amplify few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. There are three major steps (Denaturation, Annealing and Extension) in a PCR and repeat for 30 or 40 cycles.
Real-time PCR, also called quantitative real time PCR (Q-PCR/qPCR), is used to amplify and simultaneously quantify a targeted DNA molecule. It enables both detection and quantification (as absolute number of copies or relative amount when normalized to DNA input or additional normalizing genes) of one or more specific sequences in a DNA sample.
Reverse transcription PCR includes two steps. The first step is reverse transcription, in which RNA is reverse transcribed to its DNA complement (complementary DNA, or cDNA) using reverse transcriptase and primers. The second step is amplification using traditional or real-time PCR.