PCR:

          Polymerase Chain Reaction (PCR) also called ‘Molecular Phototyping’ describes the method used amplifying small and targeted DNA fragments producing millions of copies of certain genes piece. This app was launched in 1983 by Kary Mullis, a Nobel laureate 1993 for his work on PCR with Michael Smith.

Essential Components of PCR

·         Themal cyclers (thermocyclers)

·         Target DNA (DNA template)

·         Two primers(forward and reverse primers)

·         Taq polymerase (Themus aquaticus) 

·         Buffers

·         Deoxy nucleotide triphosphates (d NTP’s) 

·         Monovalent/ bivalent cation

·         Nucleotides (A/T/G/C)

·         Water

Process Involved in PCR

·         Denaturing/ melting 

·         Annealing

·         Elongation/ extension step

Different Types of PCR Techniques:

·         Nested – semi nested PCR 

·         Multiplex PCR

·         RT-PCR

·         Conventional PCR

·         Inverse PCR

·         Allele specific PCR

·         Asymmetric PCR

·         Arbitrary PCR

·         Core sample PCR

·         Degenerate PCR

·         Assembly PCR

·         Dial-out PCR

·         Digital PCR

·         Traditional PCR

·         Hot start PCR

·         In-silico PCR

·         Inter sequence PCR 

·         Ligation-mediated PCR

·         Methylation- specific PCR

·         Miniprimer PCR

·         Nano particle PCR

·         Overlap-extension PCR

·         Quantitative PCR

·         Solid phase PCR

·         Suicide PCR

·         Thermal asymmetric interplaced PCR

·         Semiquantative PCR

·         Conventional PCR

·         Colony PCR

·         After exponentional PCR

·         Standard PCR

·        Qualitative PCR

Quantitative PCR techniques

It is also referred to real time PCR. It gives an idea about how much DNA amount present in the sample. It is highly reproducible, rapid, sensitive and specific technique for automating data. Brucella species has been developed for targeting 16S23S genea by means of this technique. It has lower risk of contamination. It has 2 techniques for detection:

1. SYBER Green dye (or) fluorochromes (or) inter calculating agents

2. Taq man probes (or) fluroprobes

Qualitative PCR techniques

When PCR techniques is used for detecting a specific DNA segment, it is called as quantitative PCR method. PCR techniques are used in the identification of genes of bacteria and virus. It is fast and simple technique.

Conventional PCR

This defined as a normal PCR process. Here the primers bind specifically to each other with 2 DNA strands. Primers also limit the sequence to be replicated and a particular DNA sequence is amplified with billions of copies. The whole process takes place within 35-40 minutes repeatedly and viewed by gel electrophoresis technique.

 

Multiplex PCR Multiplex

PCR technique detects different pathogens in a single sample, used to identify exonic/ intronic sequence in specific genes. The designing of primers are different because they are meant to adhere to specific DNA sequence. This technique is used to detect viral/bacterial and other infectious agents.

 

Nested-semi nested PCR

Two sets of primers are used here for a single locus point. The first set is an amplified sequence, and the second set is complementary to the first sequence which will be shorter than the first amplified product. Nested PCR is used because it intends to reduce the contaminations in products due to the amplification of unexpected primer binding sites. Nested-semi nested PCR were used in identifying Brucella in human blood samples

Standard PCR

This is simple effiecient and sensitive technique. This method is carried out with one pair of primers which amplifies the targeted genomic sequence of Brucella species. It also helps in the early diagnosis Brucella. Standard PCR technique is used to determine the number of leucocytes DNA.

Reverse transcription PCR (RT- PCR)

Reverse transcription PCR amplification can be performed as a two-step process in a single tube or with two separate reactions. In both cases, RNA is first reverse transcribed into cDNA, which is then used as the template for PCR amplification. The primers used for cDNA synthesis can be either non–sequence-specific primers (a mixture of random hexamers or oligo-dT primers) or sequence-specific primers.

Inverse PCR

In the inverse PCR, the amplification of unknown flanking regions of DNA carried out using the known DNA sequence primers. Here, the unknown DNA sequence adjacent to the known template DNA is amplified using the primers which amplify the flanking (unknown) region near the know DNA template. With the help of the sequence information of known DNA region, the unknown flanking region of the DNA or the inserted DNA is amplified into the cyclic enzymatic reaction using the known DNA sequence-specific primers. DNA synthesis occurs outside the known DNA region. Restriction digestion is performed on the genomic DNA which digests only the unknown flanking regions but not the known DNA region.