Molecular Techniques

Question 1

Define the PCR

Answer 1

technique in which a small DNA sequence can be amplified allowing extremely small samples of DNA to be used for further testing

Question 2

What are the tools needed for a pcr

Answer 2

Thermocycler 
Template of DNA
Primer
Taq polymerase 
dNTPS for DNA synthesis

Question 3

What are the steps of the pcr

Answer 3

Denaturation at heat 94 to 98 to seperate DNA
Annealing at 50-68 to cool down sample allowing primers to to anneal to their complementary regions
Elongation at 72 to inc activity of taq polymerase

Question 4

What are the applications of the pcr

Answer 4

Realtime pcr: basic type that amplifies but also addresses need for quantification of pcr product

Reverse transcriptase: reverse transcribes RNA in to DNA then amplified by pcr to detect RNA viruses

Question 5

Define gel electrophoresis

Answer 5

Separation method based on differential of migration of charged particles in an applied electrical field

Question 6

What are tools used in gel electrophoresis

Answer 6

Chamber
Supporting material
Ethidium bromide
UV light source
DNA ladder

Question 7

What are the steps of gel electrophoresis

Answer 7

Agrose gel is poured horizontally in to electrophoresis chamber
DNA samples are pipetted in to sample wells
Application of an electric current at anodal side so DNA moves towards cathodal side
Smaller DNA fragments are at the bottom
DNA is visualized using ethidium bromide under uv light
DNA ruler is used for comparison

Question 8

What are applications of gel electrophoresis

Answer 8

Analyze results of pcr
In blotting techniques
Illness in RFLP

Question 9

Define blotting

Answer 9

Marking or staining and involve transfer of DNA,RNA or proteins on to a carrier in order to detect them

Question 10

What are the tools needed for blotting

Answer 10

Sample containing material
Tools for gel electrophoresis 
Blotting membranes(nitrocellulose)
Probes( complementary to target and marked)
Detection system

Question 11

What are the steps of blotting

Answer 11

Electrophoretic separation
Blotting
Hybridization
Detection

Question 12

What are applications of blotting

Answer 12

Southern: DNA for fingerprinting (STRs)
Northern: RNA to study mRNA
Western: proteins for antibody or antigen measurement

Question 13

What are STRs

Answer 13

Certain sequences of non coding DNA and length varies from person to person

Question 14

Define RFLP

Answer 14

Variation of DNA sequence can be recognized by restriction enzymes which cut DNA in to fragments of polymorphic lengths

Question 15

What are tools used in RFLP

Answer 15

Dna extract
Restriction enzyme
Gel electrophoresis

Question 16

What are restriction enzymes

Answer 16

Enzymes isolated from e coli and recognize double stranded DNA sequences and proceed to cut within yielding DNA restriction fragments

Question 17

What are applications of RFLP

Answer 17

For analysis of mutations based on idea that mutations can create or eliminate restriction sites

Question 18

Define dna sequencing

Answer 18

Technique that determines exact sequence of bases in a dna molecule using a dna sequencer

Question 19

What are applications of DNA sequencing

Answer 19

Human genome project

Mutation testing

Question 20

Define molecular cloning

Answer 20

Set of exp used to assemble recombinant DNA that can replicate in host organisms

Question 21

What are the tools needed for Mol cloning

Answer 21

Dna from source to be cloned
Restriction Endonucleases
Cloning vectors

Question 22

What are the steps of mol cloning

Answer 22

Restriction enzyme cuts DNA and vectors at restriction sites
Ligation of DNA fragments in to vector to form recombinant vector
Transfer in to cells
Grow host cells in culture

Question 23

What are the applications of recombinant DNA

Answer 23

Protein production such as insulin replacement
Gene therapy:inducing a normal copy of the gene that is defective in to tissues that give rise to the pathology offering a potential cure for individuals with inherited diseases