Question

a. Which laboratory procedure would a laboratory technician use to determine the

presence of a particular drug present in small quantities from a serum sample?

Discuss the principle of the procedure in full. (10)

b. Describe a procedure that one would use to construct a genomic library from DNA. (10)

Answer 1

A) Traditional method used to detect drugs abuse in laboratories was immunoassay followed by confirmation of obtained positive results by a more specific and sensitive technology gas or liquid chromatography coupled with mass spectrometry.

Immunoassay methods measure the concentration of an analyte using simple concept of antigen antibody reaction in which an antibody is raised against the specific analyte such as drugs , further antibody bind to specific antigen in the serum sample and generate a signal which can be detected using colorimetric , fluorescent or other known detection method.Cross reactivity with drugs of similar structure lead to the use of other highly specific and sensitive liquid chromatography time of flight mass spectrometry.

B) Genomic library is a collection and storage of genomic DNA in identical vectors each having different insert of DNA. They are used in sequencing of various organism.The steps involved in creation of genomic library are as follows:

1. Extraction and purification of genomic DNA from the organism: Genomic DNA is extracted and digested with restriction enzymes to generate fragments of similar size and each containing a gene. In case of Organisms having smaller genome, the fragments are separated by gel electrophoresis, excised from the gel and then can be cloned further. In case of organisms with larger genome the fragments are cloned entirely first and then clones are separated.

2.Insertion of fragments into vectors: The fragments generated are inserted into vectors which are cut by same restriction enzyme. The vector and insert are then ligated by using enzyme DNA ligase thereby creating a large of recombinant DNA molecules.

3.Trasformation in bacteria: The recombinant construct is transformed into bacteria by giving heat shock at 42 degree celsius for 90 seconds and afterwards they serve as library of genomic DNA of the organism.