molecular techniques

Question 1

purpose of PCR

Answer 1

amplify DNA from a limited source so there is sufficient ammount for analysis

Question 2

PCR components (5)

Answer 2

Template DNA, Primers in excess, Taq polymerase, deoxyribonucleotides (dNTPs) and buffer solution

Question 3

template DNA in PCR

Answer 3

contains target sequence to be amplified

Question 4

primers in PCR

Answer 4

initiaites DNA synthesis by providing free 3’ OH group for Taq polymerase to bind to and extend

2 diff primers needed to flank ends of DNA segment of interest

required in excess to increase likelilhood of binding to target sequences

Question 5

Taq polymerase in PCR

Answer 5

thermostable DNA polymerase resistant to denaturation at high temperature

Question 6

dNTPs in PCR

Answer 6

substrates for DNA replication

Question 7

buffer solution in PCR

Answer 7

contains mg2+ ions for proper dna polymerase function

Question 8

PCR STEPS (3)

Answer 8

denaturation, primer annealing, extension

Question 9

pcr denaturation

Answer 9

double stranded DNA denatures into single stranded dna by heating to 95C
breaking hydrogen bonds between comp base pairs

Question 10

pcr primer annealing

Answer 10

primer anneals specifically to 3’ end of each single stranded target DNA sequence via comp base pairing
temp lowered to 64C

Question 11

pcr extension

Answer 11

taq polymerase synthesizes complementary dna strand from free 3’ OH end of DNA primer by catalysing formation of phosphodiester bonds between dNTPs at 72C

Question 12

advantages of PCR

Answer 12

1. only small amounts of DNA required, each round of PCR number of copies of target DNA is doubled, increasig exponentially
2. use of thermostable taq polymerase allows pCR to be automated so DNA can amplify quickly

Question 13

limitations of pcr

Answer 13

1. taq polymerase lacks 3’ to 5’ proofreading ability, compounding errors
2. knowledge of sequences flanking target seq is required to design the right primers
3. taq polymerase falls off the dna template before extension ends, limits size of dna fragment to be amplified
4. minute amounts of contaminant DNA exponentially amplified along with target DNA, affects reliability of results

Question 14

gel electrophoresis

Answer 14

separates DNA based on fragment size

Question 15

wht does gel electrophoresis help with?

Answer 15

estimation of fragment size and amount of DNA

Question 16

main principles that allow gel electrophoresis

Answer 16

negatively-charged DNA migrates out well towards positive electrode when subjected to electric field
meshwork of agarose polysacch impede movement of longer fragments

Question 17

prepare gel steps

Answer 17

• place slab of gel in buffer solution with ions, allowing electricity conduction
• mix with dense loading dye to sink DNA to bototm of well
• mix with 2 coloured dyes as visual markers to show progress of migration of DNA

Question 18

Load samples steps

Answer 18

• pipe dna into wells in gel near negative electrode
• dna ladder containing dna fragments of known sizes is run in one lane to compare fragments of unknown lengths

Question 19

electrophoresis last steps

Answer 19

• negatively charged dna moves towards positive electrode when subjected to current
• gel matrix made of meshwork of polysaccharides which impedes moevment of longer fragments more than short ones
• gel treated, staining dye binds to Dna and fluoresces under UV light, used to visualise bands

Question 20

Southern blotting purpose

Answer 20

detect and confirm specific nucleotide sequences within DNA sample

Question 21

first three steps southern blotting

Answer 21

1. gel slab with dna fragments placed on top of a sponge in alkaline solution , nitrocellulose membrane placed on top of gel slab, and paper towels plus heavy weight
2. absorbent paper towels draw solution towards themselves, alkaline solution denatures double stranded dna fragments into single stranded dna
3. single stranded DNA drawn upwards onto nitrocellulose membrane and bind to membrane

Question 22

last three steps southern blotting

Answer 22

4. membrane is removed and incubated with single-stranded radioactive DNA probe, which hybridises via comp base pairing to part of the target sequence
5. excess unhybridised probes washed off
6. autoradiography performed by placing x ray film over membrane, exposing radioactive regions since probes are radioactive

Question 23

RFLP stands for?

Answer 23

restriction fragment length polymorphisms

Question 24

what are RFLPs

Answer 24

unique banding patterns when DNA is digested by restriction enzymes and separated by electrophoresis

Question 25

why do rflps arise?

Answer 25

polymorphic nature of DNA meand variations in 1. number/location of restriction sites 2. number of tandemly repeated nucleotide sequences results in unique banding patterns

Question 26

use of rflp analysis

Answer 26

disease detection, sickle cell anemia or dna fingerprinting

Question 27

disease detection with rflp

Answer 27

sickle cell anemia caused by point mutation, that alters dna sequence recognised by restriction enzyme in disease allele : MstII restriction site eliminated in normal allele: Mstll restriction site retained

Question 28

dna fingerprinting with rflp

Answer 28

detecting varying number of tandem repeats (no two humans have the same genome!)

Question 29

protocol for dna fingerprinting

Answer 29

RE on either side of tandem repeat loci + separation by electrophoresis + incubate with radioactive probes then autoradiography

Question 30

how to conduct rflp for sickle cell

Answer 30

restriction enzyme + gel electrophoresis + southern blotting OR PCR + restriction enzyme + gel electrophoresis