4C & D - PCR and gel electrophoresis

Question 1

polymerase chain reaction (PCR) is a

Answer 1

DNA manipulation technique that amplifies
DNA by making multiple identical copies

used when there is insufficient amount of dna for a sample

Question 2

Materials Required for PCR

Answer 2

DNA sample (to be amplified).

Taq polymerase (heat-resistant enzyme that synthesizes DNA).

Nucleotide bases (building blocks of DNA).

Primers (short DNA sequences that initiate replication).

Question 3

steps of PCR

Answer 3

denaturing (90-95)
- hydrogen bonds between bases break -> strands seperate -> single stranded DNA

annealing (50-55)
primers bind to complementary bases

Elongation (72)
- taq polymerase extends DNA from primers, synthesising new strands

-repeat. each cycle is 2n dna

Question 4

there are ___ primers that are ___

Answer 4

2 primers
forward primer - bids to template (3’ end)
reverse primer (binds to coding strand (3’ end)

Question 5

what is gel electrophoresis

Answer 5

used to seperate dna frag,ents by size using electric current after its been cut by restriction enzymes or amplified using PCR

Question 6

first step of GE

Answer 6

Loading DNA
- dna samples placed in agarose gel walls using micropipette
- standard ladder (dna of knows sizes) included for size comparison
- gel immersed in a buffer solution to conduct electricity)

Question 7

second step of GE

Answer 7

applying electric current
- negative elctrode - near wells
- dna (negatively charged due to sp backbone) moves towards positive electrode on the other end

Question 8

3 step of GE

Answer 8

smaller fragments - move faster through agarose pores -> travel further
- larger fragments - slower- stay closer to wells
- form distinct bands based on size after a few hours

Question 9

4th step of ge

Answer 9

gel stained w fluorescent dye. bands visible under uv

Question 10

how is eg used in genetic testing

Answer 10

detects mutations in genes
pcr amplifies target gene, the ge identifies allele size.

Question 11

how is eg used in dna profiling

Answer 11

uses str (short tandem repeats) which vary between individuals
- pcr amplifies str from crime scenes -> compares to suspect dna
- matching str confirms suspect

Question 12

str

Answer 12

short tandem repeats
- vary between individuals
- small sections of repeated nucleotides.
- found in non coding strands
- higher mutation rate than other dna
- hundreds of variants can be found