Pcr And Uses

Question 1

In replication what is needed to start rep on both leading and lagging strand

Answer 1

Rna primer 5’ to 3’

Question 2

Which polymerase primase adds rna primers to the strands in replication

Answer 2

Polymerase alpha

Question 3

Which polymerase adds dntps in replication to both lagging and leading

Answer 3

Polymerase delta

Question 4

What does polymerase epsilon do

Answer 4

Removes rna primers

Question 5

What does ligase do

Answer 5

Joins strands with phosphodiester bonds

Question 6

What are the SSBs called in replication

Answer 6

Replication protein A (rpa)

Question 7

Wht holds dna during replication

Answer 7

Sliding clamps

Question 8

Name all the things needed in PCR

Answer 8

Ssdna template 
Taq 
Dntps 
Buffer - 8.0 and salt 
Mg or Cl determine primer stringency 
Balanced temp between primers

Question 9

Where is the reverse and forward primer

Answer 9

Reverse is at the end of the 5’ to 3’ strand

Forward is at the front of 3’ to 5’ strand

Question 10

Why do primers help taq polymerase

Answer 10

Needs 3’ oh from the sugar to join to a phosphate

Question 11

How do you calculate how many dna copies there are

Answer 11

2 to the power of cycle numbers

Question 12

What is used to detect PCR products in agarose gel

Answer 12

Intercalating dyes (in between bp) 
They fluoresce under uv light

Question 13

Give an example of intercalatint dyes

Answer 13

Ethidium bromide (br)

Question 14

Name the 4 ways PCR is useful

Answer 14

Genetic tests
Diagnosis
Forensics
Personalised medication/ pharmacogenetics

Question 15

What is qpcr

Answer 15

Monitors the production of and amp of dna every 7 seconds via dye/probe

Question 16

Why is sybr green used in qpcr

Answer 16

Fluoresces only when dsdna is produced

Question 17

What is the Ct value

Answer 17

Cycle threshold - the cycle number where a product is detected by qpcr

Question 18

What is reverse transcriptase rt PCR used for

Answer 18

Measuring levels of gene expression (rna) and viral infections

Question 19

What is the first step of rt PCR

Answer 19

Rna is converted to cdna strand for PCR to happen

Question 20

What primer does reverse transcriptase use to convert rna to cDNA

Answer 20

DT primer which is T rich and binds to the poly A tail of mrna on the 3’ end

Question 21

Why is PCR used to manipulate dna

Answer 21

Understand gene function via deletion/Hr

Protein localisation

Question 22

What kind of primer is used when transforming cerevisiae budding yeast with PCR products

Answer 22

Hybrid primer

Has bases from yeast dna on the ends and the plasmid dna with the required gene in the middle

Question 23

How do yeast incorporate the PCR product into their genome

Answer 23

The yeast dna on ends is recognised as broken ends

This causes homologous recombination with the yeast dna and thus incorporates the middle PCR gene from a plasmid origin

Question 24

What gene is usually transformed into yeast from PCR via the homologous recombination

Answer 24

G418 resistance

Question 25

How is PCR used to localise proteins

Answer 25

Amplify gfp or other reporter proteins

Question 26

What does genotyping patients using PCR mean

Answer 26

Identifying which alleles they have for genes

Question 27

Why is fenotyping patients important

Answer 27

Identify carriers
Pharmacogenetics
HLA tissue typing

Question 28

Name the 2 techniques used to genotype patients using PCR

Answer 28

PCR rflp

Arms PCR

Question 29

How is PCR rflp used

Answer 29

Uses RE to cleave the amplified alleles

Eg

If patient has allele 1 which is mutated with an snp, this PCR product will be cleaved and runs further on agarose gel

Question 30

How is PCR rflp used for sorsbys fundus dystrophy diagnosis

Answer 30

Sorsbys fundus dystrophy is a mutation in timp3 gene which introduces a stop codon prematurely (RE site)

If the PCR product runs faster on agarose gel, they have an allele for sorsbys fundus dystrophy

Question 31

What is the limitation of PCR rflp

Answer 31

RE are expensive

Only works on known RE sites

Question 32

How does arms PCR detect allele base variations / mutations

Answer 32

Via allele specific primers

Question 33

How does arms PCR work

Answer 33

2 types of primers are produced 1 for allele 1 without a mutation or a primer with a known mutation in it

If the PCR product is amplified with a mutated primer, this means the person carries an allele for the mutation

Question 34

What disease is arms PCR used in

Answer 34

The f508 deletion mutation in CFTR

Question 35

Which arms PCR uses non specific allele primers

Answer 35

Tetra arms PCR

Question 36

Why is it important to genotype pathogens via PCR

Answer 36

Detect strain specifically

Better personalised medicine

Question 37

Why is PCR better at genotyping pathogens than microscopy

Answer 37

Microscopy needs a large sample of pathogen, PCR is sensitive to 1 dna molecule

Microscopy is hard to distinguish strains, PCR is specific

Question 38

Why is PCR preferred over culturing for genotyping pathogens

Answer 38

Not every strain can be cultured and it takes weeks

Question 39

What is the advantage of using PCR to genotype pathogens over an antibody response measurement

Answer 39

Not all pathogens produce a antibody response

Question 40

What would a stronger line on agarose suggest

Answer 40

Bigger infection

Question 41

What 3 things does PCR tell you about pathogen

Answer 41

If it has a resistance mutation
What strain it is
The presence of it

Question 42

How is PCR used to phenotype the disease (measure progression)

Answer 42

Measures levels of rna via reverse transcriptase PCR and qpcr is used

Question 43

What would a low Ct suggest about the phenotype of the disease

Answer 43

It is progressed more, more rna is present as the product is seen faster