PCR

Question 1

Polymerase chain reaction

Answer 1

Used to make many copies of a specific DNA region in vitro

Question 2

DNA polymerase

Answer 2

Reads template DNA and adds complementary nucleotides in 5 to 3 direction

Question 3

Taq pol

Answer 3

Has a high optimum temp and so can stay stable at high temps

Question 4

PCR components

Answer 4

DNA pol, DNA template, primers, free nucleotides, free ions and sterile water

Question 5

DNA template

Answer 5

Can be genomic or complementary DNA

Question 6

Genomic DNA

Answer 6

Very stable and it identifies mutations and genetic markers

Question 7

Complementary DNA

Answer 7

Made from RNA, it shows if a gene is being transcribed and the response of genes to treatments

Question 8

Primers

Answer 8

Short ssdna fragments that direct pol to where it needs to bind which ensures that only the region of interest is being copied

Question 9

Primer design

Answer 9

Length of 18-30 bps, gc content of 40 - 60 %, and annealing temp is 50-65°C

Question 10

What to avoid in primer design

Answer 10

4 + runs of the same base, repeat pattern of 2 bases, self-complementary primer and homology of forward and reverse primers

Question 11

Annealing temp

Answer 11

Temp at which primer duplexes with ss DNA

Question 12

Mispriming

Answer 12

When the primer anneals to an unintended location

Question 13

Free nucleotides

Answer 13

Deoxyribonucleotide triphosphates that are recruited by DNA pol to synthesize the new strand

Question 14

Free ions

Answer 14

Maintain enzyme-substrate complex and buffer the ph

Question 15

K+

Answer 15

Provides stability for primer binding

Question 16

Mg 2+

Answer 16

Catalyse phosphodiester bond and stabilizes complex of pol + primer + template

Question 17

PCR program

Answer 17

Initial denaturation, denaturation, annealing, extension, final extension and cooling for 25 - 35 cycles

Question 18

Denaturation

Answer 18

Separation of supercoiled DNA by breaking h-bonds at 95°C

Question 19

Annealing

Answer 19

Primers bind to template at 50 - 65 °C

Question 20

Extension

Answer 20

Taq pol extends primers and the new strand is synthesized at 68 - 72°C

Question 21

Thermocyclers

Answer 21

Carries out PCR because it can be programmed to run at different temps

Question 22

Visualizing PCR

Answer 22

Determine DNA fragment size by gel electrophoresis then visualize the results using a dye and UV light

Question 23

Optimizing PCR

Answer 23

By adjusting primer annealing temp, annealing time, extension time, mg 2+ conc, k+ conc, amount of template or amount of pol

Question 24

High annealing temp

Answer 24

More specific primer binding

Question 25

Mg 2+ conc

Answer 25

Adjusting it affects the number of bands produced

Question 26

Conventional PCR

Answer 26

Non-quantifiable amplification of g DNA that results in a single product

Question 27

Reverse transcriptase PCR

Answer 27

Convert RNA into cDNA, it is semi-quantitative and used to determine if the mRNA transcript is present

Question 28

Quantitative real time PCR

Answer 28

Amplification and quantification of cDNA and gene expression by quantifying the fluorescent signal produced during PCR

Low cycle number indicates a high transcript amount

Question 29

Microarrays

Answer 29

Detects thousands of genes simultaneously and investigates mutations in genes and determines if they’re on or off