BT301 Current Solved Papers 2019

Q1. Mutagenesis and its types?

Mutagenesis is a process to change the genetic information of an organism  It can occur naturally, or as a result of exposure to mutagens or induced experimentally in laboratory, Three different methods of site-directed mutagenesis have been devised i). Cassette mutagenesis ii). Primer extension iii). Procedures based on PCR.

Cassette mutagenesis

 A synthetic DNA fragment containing the desired mutant sequence is used to replace the corresponding sequence in the wild-type gene.

Primer extension: the single primer method  It involves priming in vitro DNA synthesis with a chemically synthesized oligonucleotide (7-20 bp) that carries a base mismatch with the complementary sequence.

PCR methods for site directed mutagenesis

 Single bases mismatched between the amplification primer and the template become incorporated into the template sequence as a result of amplification.

DNA sequencing: Benefits and Applications

DNA sequence information is a prerequisite for planning any substantial manipulation of the DNA Information is useful to various fields i.e. molecular & evolutionary biology, metagenomics, medicine, forensics etc.

Techniques for DNA sequencing became available in the late 1970s

Maxam-Gilbert method:

This method for DNA sequencing makes use of chemical reagents to bring about base-specific cleavage of the DNA

Q3. RT-PCR and Multiplex PCR?

RT-PCR:

Reverse transcription followed by the PCR (RTPCR) leads to the amplification of RNA sequences in cDNA form

•  RT-PCR is a sensitive means for detecting, quantifying and cloning specific cDNA molecules. Major screening strategies involve

1. . Genetic methods
2. . Sequence-dependent screening
3. . Screening expression libraries

Multiplex PCR:

Multiplex PCR is used to amplify several different DNA sequences simultaneously. Multiplex PCR is a widespread molecular biology technique for amplification of multiple targets in a single PCR experiment. In a multiplexing assay, more than one target sequence can be amplified by using multiple primer pairs in a reaction mixture. As an extension to the practical use of PCR, this technique has the potential to produce considerable savings in time and effort within the laboratory without compromising on the utility of the experiment.

real-time PCR

The main advantage of real-time PCR over PCR is that real-time PCR allows you to determine the initial number of copies of template DNA (the amplification target sequence) with accuracy and high sensitivity over a wide dynamic range.

In conventional PCR, the amplified DNA product, or amplicon, is detected in an end-point analysis. In real-time PCR, the accumulation of amplification product is measured as the reaction progresses, in real time, with product quantification after each cycle. First, amplification reactions are set up with PCR reagents and unique or custom primers. Reactions are then run in real-time PCR instruments and the collected data is analyzed by proprietary instrument software.

Q5. Disadvantage of PCR?

Setting up running required high technical skills. High equipment cost. High Test Cost High sterile environment should be provided. (DNA contamination)

Q6. Ortholog?

Orthologs are homologous genes in different organisms that encode proteins with the same function and which have evolved by direct vertical descent.

Q7. Four Step of Cloning Strategy?

Any cell based cloning procedure has four essential parts i). A method of generating DNA fragment for cloning ii). A reaction that inserts that fragment into the chosen cloning vectors iii). A means for introducing that recombinant vector into a host cell wherein it is replicated iv). A method of selecting recipient cells that have acquired the recombinant

Q8. Spotting DNA?

Spotted DNA arrays are produced by printing DNA samples on treated microscope slides. It is

where they are immobilized.

Q9. Why taq DNA polymerase use in PCR?

Taq polymerase is a thermostable DNA polymerase isolated from thermophilic bacterium Thermus aquaticus and is used in PCR to amply DNA fragments.

Q10. How Fragments Show p1258 and ps101 cs101?

Applications of microarrays

Microbial gene expression analysis

The predominate application of DNA microarray has been to measure gene expression levels

Profiling in human disease

Arrays have been used to investigate transcriptional profiles associated with human disease and to identify novel disease markers and potential new drug targets Genotyping Microarrays have been widely used as single nucleotide polymorphism (SNP) genotyping platforms