MB504T Final Term Subjective and Objective File 2023 by Sulman Ali

SUBJECTIVE&OBJECTIVE · BIO203 - Methods in Molecular Biology · Final-Term File 2023 By · SULMAN ALI (BS-ZOOLOGY) · CURRENT OBJECTIVES

1. In situ Is a polymerase chain reaction that actually takes place inside the cell on a slide.
2. In molecular Technique the term RAPD stands for Random amplified polymorphic

DNA

3. In Southern Blotting fragments are detected DNA
4. Northern blotting is used for Gene expression
5. Primary antibody used probes in Western blotting
6. How many different SNP screening technique are present. 2
7. will cut DNA only when the sequence 5’..GAATTC..3’ ECOR1
8. Enzyme responsible for joining DNA fragments DNA ligase
9. Cohesive fragment of DNA produced by digestion with EcoR1
10. Enzyme that catalyze template dependent synthesis of DNA DNA polymerase.
11. System of cutting DNA molecules was produced by Smith and Nathan.
12. Suicide PCR is typically used in Paleogenetics.
13. Which of the following Enzyme is responsible for removing phosphate group from many types of molecules including DNA? Alkaline phosphatase

Q14. Which of the following can be used to insert burnt ended foreign DNA into vector?

Ligase

15. The amount of DNA for RAPD in a reaction is 10ng
16. Mutation in DNA can be identified by Southern blot.
17. PCR _________ Polymerase chain reaction
18. RFLP________ Restriction fragment length polymorphism
19. Random Amplified Polymorphic DNA_____RAPD
20. Amplified fragment length polymorphism_________AFLP
21. which is type of microscope having beams of electrons__________ Electron

Microscope

22. Which type of pcr is done inside the cell__________In Situ PCR
23. The type of pcr which defines bacterial yeast clones is.......... Colony PCR
24. STR stands for......Short Tandem Repeats
25. How many Screening Methods SNPs have.......2
26. WGA PCR stands for....... Whole Genome Amplification Polymerase Chain Reaction
27. To amplify one strand of the original DNA more than the other, a special type of PCR is used which is known as ________.Asymmetric Pcr
28. Which is type of microscope having beams of electrons.......... Electron Microscope
29. Which type of pcr is done inside the cell...... . In Situ PCR
30. The type of pcr which defines bacterial yeast clones is.......... Colony PCR
31. STR stands for......Short Tendem Repeats
32. How many Screening Methods SNPs have.......2
33. WGA PCR stands for....... Whole Genome Amplification Polymerase Chain Reaction
34. A type of pcr which amplify single strand....... Asymmetric Pcr
35. Type of pcr which assembel many pieces into one.......... Assembly Pcr
36. Immunosorbent Electron Microscopy contains............ antibodies. Both ( monoclonal

and polyclonal)

37. In Gel Electrophoresis molecules movement depend on ......... Size
38. 11.10% DMSO
39. Works well as a........Freezing Medium
40. Antibodies recognize a unique molecule called........ Antigen
41. How many % attribute make a men unique. 0.1%
42. Denaturation temperature 94 c
43. Annealing temperature 48 to 62 c
44. Extension temperature 72 c
45. Protein of interest is detected and localized using a specific ..... Antibody
46. wet transfer is recommended for large protein.......100 kd
47. RAPD stands for ....Random amplified polymorphism DNA
48. southern blotting fragments .... DNA
49. How many different SNP screening ..... 2
50. Enzyme responsible for ............DNA Ligase
51. cohesive fragment of DNA produced .......EcoR1
52. suicide PCR is typicaly used bin ..... paleo genetics
53. How many% attribute men unique . 0.1%
54. Gel electrophoresis Molecules movement depend on size.
55. Immunosorbent Electron Microscopy contains............ antibodies. Both (monoclonal and

polyclonal)

56. STR stands short tandem repeats

CURRENT SUBJECTIVE

What are the Limitations of PCR?

1= Contamination risk 2=Primer complexities 3= Primer-binding site complexities 4= Amplifies rare species 5=Detection method

Nested PCR differ Multiplex PCR:

Nested PCR: in this type of PCR the Two pairs (instead of one pair) of PCR primers are used to amplify a fragment. First pair amplify a fragment similar to a standard PCR, and have Very low probability of nonspecific amplification.

Multiplex PCR: Multiplex PCR is a variant of PCR which enables simultaneous amplification of many targets of interest in one reaction by using more than one pair of primers. The use of multiple, unique primer sets within a single PCR reaction to produce amplicons of varying sizes specific to different DNA sequences.

RAPD PCR procedure:

Random amplified polymorphic DNA (RAPD) is a PCR based technique for identifying genetic variation. It involves the use of a single arbitrary primer in a PCR reaction, resulting in the amplification of many discrete DNA products. It does not require any specific knowledge of the DNA sequence of the target organism.

Advantages of Adenosine monophosphate deaminase (AMPD)

AMP deaminase (AMPD; EC 3.5.4.6) catalyzes hydrolysis of the amino group from the adenine ring of AMP resulting in production of inosine 5'-monophosphate (IMP) and ammonia. This reaction helps to maintain healthy cellular energetics by removing excess AMP that accumulates in energy depleted cells. Furthermore, AMPD permits the synthesis of guanine nucleotides from the larger adenylate pool. This enzyme competes with cytosolic 5'-nucleotidases (c5NT) for AMP. Adenosine, a product of c5NT is a vasodilator, antagonizes inotropic effects of catecholamines and exerts anti-platelet, anti-inflammatory and immunosuppressive activities.

Hot start PCR:

This is a technique that reduces nonspecific amplification during the initial set up stages of the PCR. This technique may be performed manually by heating the reaction components upto the melting temperature (e.g, 95°C). Before adding the polymerase, specialized enzyme systems have been developed that inhibit the polymerase's activity at ambient temperature. This is done either by the binding of an antibody or by the presence of covalently bound inhibitors that only dissociate after a high-temperature activation step. DNA PolymeraseEubacterial type I DNA polymerase, Pfu.

Differentiate between the each Step of PCR:

1= Denaturation: This is the First regular cycling event and consists of heating the reaction to 93°C - 98°C for 2045 seconds. It causes melting of DNA template yielding single strands of DNA.

2= Annealing: In this The reaction temperature is lowered to 40-65°C for 20-40 seconds. Annealing of the primers to the single stranded DNA template.

3= Extension: At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTP's. The temperature at this step depends on the type of DNA polymerase used. Taq polymerase has its optimum activity temperature at 7080°C. Commonly 72°C is used.

Applications Southern blotting:

Southern blotting technique is used to detect DNA in given sample. DNA finger printing is an example of southern blotting. Used for paternity testing, criminal identification, victim identification. To isolate and identify desire gene of interest. t is mostly used in the identification of viral infection and certain bacterial infections.

Applications of western blotting:

2= Detecting different isoforms of proteins. 2= Detecting protein–protein interactions. 3= Detecting protein–DNA interactions. 4= Detecting post-translational modification. 5= Detecting proteins' subcellular localization. Western blot is often used in research to separate and identify proteins. In this technique a mixture of proteins is separated based on molecular weight, and thus by type, through gel electrophoresis.