Molecular Bio

Question 1

What Is the function of PCR
hint: amplify

Answer 1

Polymerase Chain Reaction is used to amplify DNA from a limited source of DNA so that there is a sufficient amount for analysis

Question 2

PCR components (4)

Answer 2

Template DNA; Primers, Taq polymerase, dNTPS and Buffer

Question 3

3 step process of PCR
hint: DPE

Answer 3

Denaturation –> Primer Annealing –> Extension

Question 4

Describe Denaturation in detail
hint: DS-> SS

Answer 4

Double stranded DNA denatures into SIngle stranded DNA by heating to 95 degC as weak hydrogen bonds between complementary bases of each strand are broken due to increased molecular vibrations

Question 5

Describe primer annealing in detail
hint:CBP

Answer 5

each primer anneals specifically to the 3’ end of each single stranded target DNA sequence via complementary base pairing when the temperature is lowered to 64 degC

Question 6

Describe extension in detail
hint: taq polymerase

Answer 6

taq polymerase synthesizes the complementary DNA strand form the free 3OH’ end of the DNA primer by catalysing the formation of phosphodiester bonds between dNTPS when the temp is increased to 72 degC

Question 7

what are primers
hint: SS DNA, DNA synthesis

Answer 7

synthetic SS DNA fragment needed to initiate DNA synthesis by providing a free 3OH’ ground for taq polymerase to bind to and extend; 2 different primers are required; each is complementary to the sequence at 3’ end of each SS target DNA sequence
required in large excess

Question 8

what is taq polymerase

Answer 8

thermostable DNA polymerase –> resistant to Denaturation at high temps

Question 9

Advantages of PCR
(theres 2)
hint: amt of DNA required, automation

Answer 9

only a MINUTE AMOUNT OF DNA is required; with each round of PCR the number of copies of target DNA is doubled; thus no. of desired sequence increases exponentially and there is sufficient DNA for analysis.
use of thermostable taq polymerase allows for automation and fast amplification

Question 10

Limitations of PCR
(4)
hint: proofreading; flanking sequences, size of fragment, contaminant DNA

Answer 10

1. taq polymerase lacks 3’ to 5’ proofreading ability, hence errors will be compounded
2. knowledge of sequences flanking the target sequence is require for approp primers
3. taq polymerase tends to fall of the DNA template before chain extension is complete if the strand is too long. hence there is a limit to the size of DNA fragment to be amplified.
4. Minute amts of contaminant DNA can be amplified and hence affect the reliability of the results

Question 11

Gel electrophoresis basic principle

Answer 11

separates DNA based on fragment size

Question 12

first 3 Steps of AGE –> 12 in total
hints: buffer
dye
colour

Answer 12

1. slag of agarose gel is placed in a a buffer solution containing ions which allow the conduction of electricity when the current is turned on
2. the dna sample is mixed with a dense loading dye containing glycerol and 2 coloured dyes. glycerol makes the DNA sample denser than the buffer solution so that the DNA sample can sink to the bottom of the well.
3. since DNA is invisible, the dyes colour the DNA sample and will indicate if the DNA has been loaded correctly into the well.

Question 13

steps 4-6 of AGE
hint: all about the dyes + where are the DNA samples pipetted?

Answer 13

4.a dye often runs ahead of the DNA sample and gives an indication of when electrophoresis must be stopped so that the samples do no run out of the gel. the other dye gives an indication of the position of the larger fragments on the gel.
5. the two coloured des act as visual markers which help to monitor the progress of the migration of the invisible DNA fragments.
6. DNA samples are pipetted into the well in the gel near the negative electrode.

Question 14

steps 7-9 of AGE
hint: comparison of fragments; DNA charge? anode or cathode?; what is the gel made of?

Answer 14

7. A DNA ladder which contains DNA fragments of known sizes is run in one of the lanes and acts as a standard for which to compare fragments of unkown size.
8. Negatively charged DNA moves towards the positive electrode (anode when subjected to an electric current.
9. the agarose gel matrix made of a meshwork of polysaccharides which impedes movement of longer fragments more than shorter fragments. the longer fragments thus migrate more slowly than the shorter fragments.

Question 15

steps 10-12 of AGE
hint: when to stop?; what is ethidium bromide?; what information do u get from this test?

Answer 15

10.before the loading dye reaches the end of the gel, the current is turned off
11. to visualize the bands, the gel can be treated with a staining dye that binds DNA (e.g ethidium bromide) and fluoresces under UV light
12. the fragment size can be estimated based on position of the band relative to markers and the amt of DNA based on intensity and thickness of the band

Question 16

wtf is southern blotting

Answer 16

a tool to detect specific nucleotide sequences within a sample

Question 17

steps 1-3 of southern blotting
hint: nitrocellulose membrane + ph14, DS->SS; binds to ?

Answer 17

1. gel slab is placed on top of the sponge and under a nitrocellulose membrane . a stack of paper towels are placed on top of the nitrocellulose membrane. these are placed in a tray of alkaline solution. a heavy weight is placed above the paper towels.
2. absorbent paper towels draw the solution towards themselves and the alkaline solution denatures DS dna to SS dna
   3.SS DNA on the gel is then drawn upwards onto the nitrocellulose membrane and binds to the membrane.

Question 18

steps 4-6 of southern blotting
Hint: radioactive probe?; wash off; autoradiography?

Answer 18

4. nitrocellulose membrane is removed and incubated with SS, radioactive DNA probe which hybridises via complementary base pairing to part of the target sequence..
5. the excess unhybridised probes are washed off.
6. Autoradiography is performed, placing X -ray film over the membrane. radioactive regions exposes the film, forming an image that correspond to the bands that have base-paired with probe.

Question 19

WTF IS AN RFLP
-HINT BANDING PATTERN

Answer 19

restriction fragment length polymorphisms
- unique banding patterns amongst individuals when their dna is digested by restriction enzymes and separated by gel electrophoresis

Question 20

why do RFLPS arise? where will there be variations in?
HINT: poly what? lokasi/numero

Answer 20

1`. polymorphic nature of DNA
2. variation in the number/location of restriction sides + the number of tandemly repeated nucleotide sequences

Question 21

SICKLE CELL ANAEMIA RANDOM BULLSHIT GO
hint: what kind of polymorphism; what kind of mutation and where is the polymorphism found?

Answer 21

DNA polymorphism in SCA is a single nucleotide polymorphism(SNP). there is a difference in a single base pair due to a pt mutation. the SNP is within the coding region

Question 22

Use of RFLP analysis in disease detection e.g sickle cell anaemia
Hint: restriction site diff?; DNA fragments show what difference?

Answer 22

A difference in a single nucleotide can result in a gain OR loss of a restriction site of an enzyme; this when a particular section of DNA in individuals with the mutation and without the mutation is digested with the same restriction enzyme, DNA fragments of different lengths will result. Analysis of the banding pattern that arises will allow determination of the presence of disease causing allele or the normal allele.

Question 23

Restriction enzyme used in sickle-cell anaemia

Answer 23

Mst II within the beta-globin gene