Biotechnology and Bioinformatics (1+1)

2.1.2.2. DNA polymerase and reverse transcriptase

DNA polymerases synthesize complementary nucleotide sequence on a template nucleotide strand.

• DNA polymerase I, isolated from E. coli, synthesizes a complementary strand on a template DNA in 5¹ → 3¹ direction.
• It also possess low level of exonuclease activity in both 5¹ → 3¹ and 3¹ → 5¹ directions.
• This enzyme is used in labeling of DNA to prepare probe .

i) Klewnow enzyme is the large fragment of the DNA Polymerase I of E. coli.

• It possess 5¹ → 3¹polymerase activity and 3¹ →5¹ exon uclease activity but lacks the exonuclease activity in 5¹ → 3¹ direction.

ii) Tag DNA polymerase is isolated from a bacterium Thermus aquaticus, living in hot springs and active even at 94ºC.

• This enzyme is highly thermostable for which it is used for DNA amplification during polymerase chain Reaction (PCR) . It does not have 3¹→ 5¹ exonuclease activity and hence cannot carry out proof reading.

Reverse Transcription of mRNA

Recently, a superior method of selecting desired gene s has been discovered, which is called reverse transcription of mRNA.

• The desired protein is first refined and purified, and next it is administered to a rabbit to stimulate the synthesis of an antibody against it.
• Antibody formation is a natural defense mechanism. When pathogenic bacteria or other extraneous substances invade our bodies, we recognize them as foreign and produce specific proteins which can bind to them, leading to inactivation or destruction of the invader.
• These binding proteins are called antibodies. Antibody specificity for antigens is extremely high.

Following this step, the antibodies produced in the rabbit are mixed with homogenized cells in which the desired protein is being synthesized. The antibodies specifically bind to the proteins while in the process of being synthesized.

• The anti-body protein-m-RNA ribosome complex sediments and m-RNA is extracted from this sediment.
• Next, the m-RNA is mixed with the enzyme “reverse transcriptase”. (This enzyme was found in some viruses having RNA as genetic information instead of DNA. The virus utilizes reverse transcriptase to catalyze the reverse process of synthesizing a complementary DNA chain on an RNA template).
• The genes specifying the desired protein can be obtained by using reverse transcriptase to make a complementary single-stranded DNA molecule synthesized on the m-RNA template.
• Then this single stranded DNA is converted into a double-stranded complementary DNA molecule by using the enzyme DNA polymerase.