Molecular tech. DNA analysis

Question 1

What is PCR?

Answer 1

(Polymerase chain rxn)
Amplifies SPECIFIC sequence of DNA (short) from small original sample
IN VITRO (outside organism) w thermal cycler in centrifuge

Question 2

Stage 1 of PCR

Answer 2

Denaturation of DNA strands 95°C

Heat DNA mixture separate dsDNA into 2 complementary ssDNA

Breaking H bonds betw CBP

Question 3

Stage 2 of PCR

Answer 3

Annealing of primers 55°C

Cool DNA mixture to 55°C, complementary DNA primers anneal to 3’ ends of ssDNA template

Excess primers -> prevent from reannealing

Question 4

Stage 3 of PCR

Answer 4

Primer Extension 72°C
Heat to 72°C optimum temp for 5’ to 3’ direction elongation
Primers provide 3’OH (Taq polymerase to add Deoxyribonucleotides to 3’)
Replicated hence 2 copies of DNA molecule
Cycle repeated 30-40 times (exponential increase,doubles every cycle)

Question 5

Benefits of PCR

Answer 5

1. Only minute DNA needed for PCR
2. Amplifies large qty w/o cells
3. Faster & more efficient, exponential increase
4. Very specific due primers for desired DNA sequence to be copied (only anneal to 3’ end flanking target DNA sequence

Question 6

Disadvantages of PCR

Answer 6

1. PCR sensitive, contamination -> unwanted sequences
2. Sequence flanking target DNA needs to be known for suitable DNA primers
3. Taq DNA polymerase low rep fidelity lacks 3’ to 5’ EXONUCLEASE proofreading activity
4. Standard PCR only copies up to 25k bases

Question 7

Function of Gel Electrophoresis

Answer 7

To seperate nucleic acids according to molecular size

Before gel e ->
Isolate DNA sequence either
PCR (amplify target sequence w specific primer)
Restriction enzyme (Total DNA hydrolysed into fragments by R enzymes)

1. Mix DNA samples w loading dye containing bromophenol blue & glycerol
2. Add DNA ladder soln in separate well reference est. size
3. DNA molecules from -ve cathode goes to +ve anode
4. Longer fragments slower, shorter smaller fragments faster, further

Question 8

How to visualise specific bands in gel e

Answer 8

Carry out southern blotting, & nucleic acid hybridisation

Autography for radioactive labelled probe
UV light for fluorescent labelled probe

Question 9

How to visualise all bands in gel e

Answer 9

Add ethidium bromide
UV light to visualise

Question 10

Function of Southern blotting & NAH

Answer 10

SB: Transfer DNA fragments from agarose gel to nitrocellulose membrane, gel breaks easily, can be reused

NAH: To detect specific DNA fragment w radioactive/fluorescent probe

Question 11

How to carry out SB & NAH

Answer 11

1. Transfer DNA fragments from gel to nitrocellulose membrane by capillary action (form replica of gel)
2. Denature dsDNA to ssDNA by NaOH, breaks H bonds betw CBPs
3. Incubate w radioactive/fluorescent probe (DNA/RNA), complementary to target DNA sequence & binds via H bond