Molecular Diagnostic Techniques In The Lab

Question 1

Molecular biology includes the manipulation of:

DNA
RNA
Protein synthesis

True or false

Answer 1

True

Question 2

DNA of a normal human cell contains how many genes❓

Answer 2

30000-120000 genes

Question 3

Exposure of DNA just below the boiling point (90-96C) causes it to become denatured

True or false

Answer 3

True

Question 4

What do restriction endonucleases do in molecular diagnostics❓

List a few examples

Answer 4

They break DNA strands into oligonucleotides for easy analysis

EcoRI
HindIII
HeaIII

Question 5

What is the function of ligases in molecular diagnostics❓

What procedures are they useful for❓

Answer 5

They seal the sticky ends of DNA together by rejoining the phosphate-sugar bonds cut by endonuclease

Recombinant DNA
Hybridization

Question 6

What is the function of reverse transcriptase in molecular diagnostics❓

What procedures are they useful for❓

Answer 6

Converts RNA to DNA

Replication of AIDS and other retroviruses

Question 7

Southern blotting…

1. Is used to❓
2. The DNA is digested with ______ to obtain DNA fragments❓
3. Digested DNA is arrayed by size using ____ in a _____ gel

Answer 7

Used to analyze a gene at the level of genomic DNA

Restriction endonuclease

Electrophoresis, Agarose gel

Question 8

Enzymatic chain-termination

1. is a method used in❓
2. What enzyme does it depend on❓
3. Chained formed from this process are resolved using ____ in a _____ gel
4. What are the applications this procedure❓

Answer 8

DNA sequencing

DNA polymerase

Electrophoresis, urea-containing polyarylamine gel

Antibiotic resistance study
ART resistance monitoring
Identifying microbes
Monitoring cancer treatment

Question 9

Recombinant DNA Molecular biology is primarily used for❓

Practical applications❓

Answer 9

Remove genetic material from one organism and combine it with that of
another organism

Genetically manipulating bacteria to produce hormones, enzymes etc

Question 10

What is hybridization❓

What is it’s use❓

Answer 10

Production of offspring from 2 genetically different parents.

Union of 2 different nucleic acids at their complementary sites

To produce gene probes (detect specific nucleotide sequences in unknown samples)

Question 11

What is northern blotting used for❓

What does it depend on❓

Chained formed from this process are resolved using ____ in a _____ gel

What is it’s application❓

Answer 11

Used to detect specific RNA sequences in a specimen

A denaturing agent ie formaldehyde/gloxal/DMSO

Electrophoresis, denaturing agarose gel
⬇️
Nitrocellulose/nylon based membrane

Demonstration of oncogenes in some human cancers

Question 12

PCR is a process that was developed by❓

Using a❓

What is it used for❓

What does it require❓

What are the three steps❓

Answer 12

Karry Mullis, 1985

Thermocycler

Genetic photocoping

Template molecule
Primer (synthetic oligonucleotide)

Denaturation
Annealing
Elongation

Question 13

Denaturation is done at what temperature?

What events occur here❓

Answer 13

94-96C

Double stranded DNA break up in two separate parts

All enzymatic rxns stop

Question 14

Annealing/Hybridization is done at what temperature?

What events occur here❓

Answer 14

54C

Hydrogen bonds are constantly formed btwn ss primers and template

Polymerase starts copying the template from 3prime end

Question 15

Elongation is done at what temperature?

What events occur here❓

Answer 15

72C

Polymerase elongates DNA

Question 16

What are the limitations of PCR?

Answer 16

Contamination of sample

Mutation of infectious agent