MB504T Final Term Past Papers by Kainat Alvi and Arshia Ashfaq

METHODS IN MOLECULAR BIOLOGY (MB504T) · CURRENT FINALTERM PAPER SPRING 2024(23July) · SHORT QUESTIONS(3) · 1. Negative control and blank reaction

NEGATIVE CONTROL: Controls for specificity of the amplification reaction contains all reagents and a DNA template lacking the target sequence.

BLANK REACTION: Controls for contamination contains all reagents except DNA template.

2. dNTPs

Deoxynucleoside triphosphates (dNTPs) are the building blocks from which the DNA polymerase synthesizes a new DNA strand during successive cycles of PCR amplification.

dNTPs consist of four basic nucleotides - dATP, dCTP, dGTP, and dTTP. The concentration of each dNTP (dATP, dCTP, dGTP, dTTP) in the PCR is usually 200 μM.

3. Give the name of six PCRs

o Standard PCR o Real-Time PCR o Reverse Transcription PCR o Nested PCR o Multiplex PCR o Hot Start PCR o Long Range PCR o Assembly PCR o Allele-Specific PCR o Colony PCR

Q4. What is Cell line?

Cell lines are cultures of animal cells that can be propagated repeatedly and sometimes indefinitely. They arise from primary cell cultures. Primary cultures are initiated directly from the cells, tissues, or organs of animals and are typically used in experiments within a few days. Cell lines are frozen to provide a source of inoculum for future cultures

5. Assembly PCR

Assembly PCR used to assemble two or more pieces of DNA into one piece.

❖ Assembly PCR Principle:

Assembly PCR is the synthesis of long DNA structures by performing PCR on a pool of long oligonucleotides with short overlapping segments, to assemble two or more pieces of DNA into one piece.

6. Which type of PCR used in amplification of DNA from fossils and how false amplification can be avoided/ Suicide PCR

control PCR reaction. Primers should always target a genomic region never amplified before in the lab using this or any other set of primers. This ensures that no contaminating DNA from previous PCR reactions is present in the lab, which could otherwise generate false positives. Use of any primers combination only once in a PCR.

7. SIGNIFICANCE OF SNPs

o In disease diagnosis o In Finding predisposition to diseases o In drug discovery & development o In drug responses o Investigation of migration patterns

8. SNP types

The SNPs in the coding region of gene are divided into two types:

SYNONYMOUS SNPS(Silent SNPs): The synonymous SNPs do not change the amino acid sequence of protein or not affect the protein function.

NON-SYNONYMOUS SNPS: SNPs with single amino acid substitution in a protein sequence that may lead to missense mutation or nonsense mutation.

o Missense SNPs: Change the amino acid sequence of the protein, potentially altering protein function. o Nonsense SNPs: Introduce a premature stop codon, leading to a truncated protein that is usually non-functional.

9. Characteristics of SNPs

SNP definition: A SNP is defined as a single base change in a DNA sequence that occurs in a significant proportion (more than 1 percent) of a large population. In human beings, 99.9 percent bases are same. Remaining 0.1 percent makes a person unique.

o SNPs are found in § coding and (mostly) noncoding regions. o Occur with a very high frequency § about 1 in 1000 bases to 1 in 100 to 300 bases. o The abundance of SNPs and the ease with which they can be measured make these genetic variations significant. o SNPs close to particular gene acts as a marker for that gene. o SNPs in coding regions may alter the protein structure made by that coding region.

10.

Two enzyme and methods for vitro molecules joining

Mainly three methods are used for joining DNA in vitro

i. Joining covalently annealed cohesive ends by DNA ligase ii. Joining blunt-ended fragments by DNA ligase from phage T4 infected E. coli iii. The third utilizes the enzyme terminal deoxynucleotidyltransferase to synthesize homopolymeric 3’ single stranded tails at the ends of fragments.

ENZYME NAMES:

i. DNA Ligase ii. Alkaline Phosphatase

11.

DNA ligase

An enzyme that creates a phosphodiester bond between the 3’ end of one DNA segment and the 5’ of another. E. coli and phage T4 encode enzyme, DNA ligase, which seals single stranded nicks between adjacent nucleotides in a duplex DNA chain. DNA ligase can repair the nicks produced after the association of cohesive ends of DNA strands

12.

Role of phosphate removal during DNA cohesive end joining

Alkaline Phosphatase:

An enzyme responsible for removing phosphate groups from many types of molecules including DNA. Treatment of linearized plasmid vector DNA with alkaline phosphatase to remove 5’- terminal phosphate groups will prevent recircularization and plasmid dimer formation.

13.

Difference between adapter and linker

ADAPTORS: Chemically synthesized adaptor molecules which have a preformed cohesive end can be used to insert blunt ended foreign DNA into vector. OR Adapters are added to the ends of DNA fragments to provide specific sequences for ligation or PCR priming.

LINKER: In contrast, linkers are inserted between two DNA fragments to connect them, providing flexibility.

The main difference between linker and adaptor is that former having blunt ends while later have one cohesive end. 14.

Which method is used for detect polymorphism of DNA

AFLP PCR : AFLP is a highly sensitive PCR-based method for detecting polymorphisms in DNA. AFLP can be also used for genotyping individuals for a large number of loci.

15. On what basis are proteins separated in native gel electrophoresis?/Making protein on native gel electrophoresis on the base of which factor? asa hi kuch question tha

In native gel electrophoresis, proteins are separated based on their native properties, including charge, size, and shape. The technique preserves the protein’s natural structure, allowing for separation according to their inherent characteristics, with proteins having a higher charge, smaller size, or more compact shape migrating faster through the gel. 16. Uses/Applications of PCR in Clinical Microbiology.

o Identification of bacteria:

Slow growing bacteria Bacterial antibiotic resistance genes. o VIRUS: almost all viruses identification and viral load.

o FUNGUS:

Aspergillus spp Candida spp Plasmodium spp Trypanosoma spp Leishmania spp Babesia spp

17. Uses of PCR in forensic science.

Forensic science is the application of scientific procedures to solve criminal and legal matters. Molecular methods are used to established the filiations of a person or to obtain evidence from minimal samples of saliva, semen or other tissues

18. DNA profiling.

DNA profiling or DNA fingerprinting is a forensic technique used to identify individuals by characteristics of their DNA.

19.

Western Blotting and uses/Applications

A technique used to detect the presence of a specific protein in a complex protein mixture.

Principle: Western blotting separates proteins by size, transfers them to a membrane, and detects specific proteins using antibodies, allowing for identification and quantification of individual proteins in a complex mixture.

❖ APPLICATIONS/USES:

o Analysis Of IgG Fractions purified from human plasma. o Diagnosis of HIV by ELISA, involves the western blotting technique. Western blotting technique is also used to Detect Some Forms Of Lyme Disease. o Western blotting technique is used in Definitive Test For BSE, which is commonly know as Mad cow disease. o Confirmatory Test For Hepatitis-B involves western blotting technique. o Western blotting test is used in the Analysis Of Biomarkers such as hormones, growth factors & cytokines. o This technique is also employed in The Gene Expression Studies

20.

Limitations in Western Blotting

o very delicate and time consuming process. A minute imbalance at any level of the procedure can skew the results of the entire process. o Incorrect labeling of the protein can happen due to the reaction of secondary antibody. o Cause erroneous in bands or no bands due to insufficient transfer. o Well trained technicians are required for this technique. Primary antibody availability is crucial. o It is just a semi-quantitative at best. Only an approx. estimation & not a precise measurement of molecular weight of the protein is possible

21.

Western Blotting And Who It Use?

Western blotting is a laboratory technique used to detect and analyze proteins in a sample, commonly used by research scientists, clinical researchers, biotechnology companies, and forensic scientists to identify, quantify, and characterize proteins through gel electrophoresis, transfer, probing with antibodies, and visualization.