Question

Describe the principles behind and the applications of the following:

a) Reverse transcriptase-PCR b) Cloning DNA into a plasmid vector

c) SDS-PAGE

d) Restriction mapping

e) Sanger Sequencing of DNA

Note

Detail explanation is required for each .

Answer 1

a) Reverse transcriptase - polymerase chain reaction (RT - PCR) is most reliable molecular biology method for detection of RNA expression. In this method, The RNA is converted to complentary DNA (cDNA) using reverse transcriptase enzyme. RT-PCR helps to determine the RNA expression qualitatively. In RT-PCR the cloned genes expression is studied by producing cDNA from RNA using reverse transcriptase and further the DNA is amplified into multiple copies using traditional PCR. RT-PCR can also be used for quantification of RNA, in both relative and absolute terms by combining the q- PCR (quantitative PCR).

b) Cloning vector is defined a short fragment of DNA of known size in which the gene of interest can be inserted and is introduced into an organism for transformation of the desired gene. The cloning vectors include plasmids of bacteria or yeast and viral vectors that allow a gene to be inserted and enable introducin into anoter organism. The newly introduced gene in an organism is transformed into its DNA and is amplified by DNA replication and is expressed. For example, a gene encoding a specific protein is introduced into a plant through a plasmid vector. In the plant cell, the new gene is incorporated into plant cells' DNA and further replicated and expressed to produce the specific protein in plant cells.

c) SDS-PAGE: Sodium Dodecylsulfate - Polyacrylamide Gel electrophoresis is analytical technique used to seperate the proteins basing on their molecular weight. SDS is an anionic detergent that disrupts the disulfide bonds of the protein molecules resulting in free proteins from three dimesional folding. The protein molecules on electric field migrate according to their size and small proteins migrate faster than large proteins. This is an accurate method to determine the size, molecular weight and concentration of proteins.

d) The DNA sequences contain special and marked nucleotide sites that can be cleaved into fragments by restriction endonuclease enzymes. These restriction enzymes cut the DNA producing short fragments that can be used for cloning experiments or arrange DNA in sequence. In restriction mapping, the entrie DNA sequence can be known.