BT301 Final Term Past Papers Set 2

MUHAMMAD IMRAN · Bt301 Final Term Paper 2019 · 1. Orthology.2

Orthologs are homologous genes in different organisms that encode proteins with the same function and which have evolved by direct vertical descent.. Orthologs are genes in different species that evolved from a common ancestral gene by speciation, and, in general, orthologs retain the same function during the course of evolution. Identification of orthologs is a critical process for reliable prediction of gene function in newly sequenced genomes.

Q2. Write principle of spotted DNA array... (3 marks)

Spotted DNA arrays are produced by printing DNA samples on treated microscope slides. It is made by transferring or spotting DNA clones or PCR product individually onto a solid support where they are immobilized.

3. Write down the main features of Real-time PCR and multiplex PCR? 5

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

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.

Multiplex PCR:

Multiplex PCR is used to amplify several different DNA sequences simultaneously.

4.

PCR, types and applications? 10

PCR: Polymerase chain reaction, or PCR, is a laboratory technique used to make multiple copies of a segment of DNA. PCR is very precise and can be used to amplify, or copy, a specific DNA target from a mixture of DNA molecules.

Types: Nested PCR, RT-PCR, qPCR, Multiplex PCR.

PCR has widespread applications in various fields of life sciences including genetic engineering, medical, forensic, agriculture, environment etc.

PCR-Gene cloning and expression

PCR has been used in gene cloning and screening of genomic libraries.

PCR-Medicine:

PCR has major impact on medicine especially in the field of clinical microbiology/diagnosis Molecular tools have also allowed performing prenatal genetic diagnosis.

PCR-Forensic sciences:

Forensic science is the application of scientific procedures to solve criminal & legal matters Molecular methods are used to establish the filiations of a person or to obtain evidence from minimal samples of saliva, semen or other tissues. PCR-DNA profiling: DNA profiling or DNA fingerprinting is a forensic technique used to identify individuals by characteristics of their DNA

PCR-Agricultural sciences and environment

PCR has also facilitated research in detection of pathogens in plants, animals and environment.

PCR-Molecular paleontology

Molecular paleontology refers to the recovery and analysis of DNA and protein from ancient human, animal and plant remains.

5. Applications of PCR? 3

PCR has widespread applications in various fields of life sciences including genetic engineering, medical, forensic, agriculture, environment etc.

6. DNA sequencing: Benefits and Applications 5

DNA Sequence: DNA sequence information is a prerequisite for planning any substantial manipulation of the DNA

 Information is useful to various fields i.e. molecular and evolutionary biology, metagenomics, medicine, forensics etc. Techniques for DNA sequencing became available in the late 1970s . DNA sequencing is the process of determining the precise order of nucleotides within a DNA molecule. It includes any method or technology that is used to determine the order of the four bases—adenine, guanine, cytosine, and thymine—in a strand of DNA. The advent of rapid DNA sequencing methods has greatly accelerated biological and medical research and discovery.

Q7. type of Resistance observed in p1258 plasmid of staphylococcus (2)?

Expression of Staphylococcus plasmid genes in E. coli pSC101 contain Tetracycline resistance P1248 from Staphylococcus

8. What is epitope? 2

the part of an antigen molecule to which an antibody attaches itself a molecular region on the surface of an antigen capable of eliciting an immune response and of combining with the specific antibody produced by such a response. Immunochemical screening involves the use of antibodies that specifically recognize antigenic determinants on the polypeptides synthesized by a target clone

 The molecular target for recognition is generally an epitope

9. Why taq DNA polymerase use in PCR? 2

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

10. What is the strategy used to synthesis the second cDNA from first strand of cloning? 3

In nested PCR, two sets of primers are used in two successive PCRs First set of primers allows a first amplification

Product of first PCR is subjected to a second PCR using the second set of primers.

Q11. Describe the importance of plasmid copy number and stability (3 marks)

The effect of plasmid copy number

The number of ribosomes in a cell far exceeds any one class of mRNA. One way of increasing the expression of a cloned gene is to increase the number of the corresponding transcript

Plasmid stability:

Having maximized the expression of a particular gene it is important to consider what effects this will have on the bacterium harboring the recombinant plasmid. The loss of plasmids due to defective partitioning is called segregative instability. Structural instability of plasmids may arise by deletions or rearrangements of DNA.

12. · Mutagenesis and its types?10. · Mutagenesis:

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. After E. coli transformation, heteroduplex that are formed are either that of original wild type DNA or that containing the mutated base.

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. In megaprimer method, the product of the first PCR is used for the second PCR.The advantage of a PCR-based mutagenic protocol is that the desired mutation is obtained with 100% efficiency.

Q13. Disadvantage of PCR?

Q14. Four Step of Cloning Strategy?

Any cell based cloning procedure has four essential parts