PCR

Question 1

why is PCR useful?

Answer 1

removes the need for cloning vectors or transformation as able to amplify the amount of DNA in a short amount of time

Question 2

what things are required for a PCR?

Answer 2

source DNA, supply of nucleotides, heat resistant polymerase, primers for DNA synthesis

Question 3

what are oligonucleotides?

Answer 3

DNA sequences of only 10-20 bps

Question 4

what do the DNA primers do?

Answer 4

complementary to the 3’ end and initiate DNA synthesis and ‘flank’ the sequence

Question 5

what is Taq polymerase?

Answer 5

heat stable DNA polymerase

Question 6

where was Taq polymerase found?

Answer 6

isolated from a bacterium that lives in hot springs

Question 7

what is pfu polymerase?

Answer 7

a more accurate and stable heat resistant polymerase yet more expensive

Question 8

how many cycles usually occur in PCR?

Answer 8

30-40 cycles

Question 9

what occurs in denaturation?

Answer 9

reaction mixture heated to 94 degrees causing the DNA to become ss

Question 10

what occurs in annealing?

Answer 10

temperature reduced to 54-60 degrees to allow annealing of primers

Question 11

what happens in extension?

Answer 11

temperature raised to 72 degrees so the polymerase begins to add bases and both strands are copied

Question 12

why is an extra 5 minutes allowed at the end of the final cycle?

Answer 12

to allow for any completion for any unfinished template strands

Question 13

what causes the synthesised strands to be too long?

Answer 13

polymerase has no stop signals

Question 14

when do the strands become to be exact?

Answer 14

after 3 cycles

Question 15

why does PCR not fully replace gene cloning?

Answer 15

some errors in cloning so instead PCR amplifies the DNA to then be inserted into a vector

Question 16

how can PCR strands used for implant?

Answer 16

primers can be used to add restriction sites into the DNA so they can be cleaved

Question 17

what are the advantages of PCR?

Answer 17

only need a small amount of DNA, variety of sources, can detect genetic disorders, only need around 20 cells

Question 18

what kinds of regions are used for PCR in fingerprinting?

Answer 18

a highly polymorphic region so that no two individuals will have the same sized fragments

Question 19

how are microarrays prepared?

Answer 19

make cDNA which is fluorescently labelled and apply to the array with each gene in a different well

Question 20

how can different tissue states be tested on the same array?

Answer 20

use a different coloured label for different tissues

Question 21

what is RNA sequencing?

Answer 21

cDNA is made from mRNA and is then sequenced and then mapped by computer into a genome sequence

Question 22

what does cas-9 protein do?

Answer 22

acts with a guide strand of RNA that has been made by CRISPR

Question 23

what does CRISPR do when the guide strand enters?

Answer 23

cuts both strands of the DNA that is complementary to the guide RNA

Question 24

what causes the gene to be inactivated?

Answer 24

when the DNA is repaired, nucleotides may be introduced or removed causing deactivation

Question 25

how can mutated genes be repaired using CRISPR?

Answer 25

by introducing a segment of the wild type gene and using this as a template

Question 26

what is gene therapy?

Answer 26

affected cells are altered by the deletion of addition of a specific gene

Question 27

how is gene therapy carried out?

Answer 27

a genetically engineered virus is used to infect malignant cells with a normal version of the gene

Question 28

what are the issues for gene therapy?

Answer 28

ethical issues, placement of the gene and control of gene expression

Question 29

what conditions need to be met for gene therapy?

Answer 29

serious and incurable disease, needed lifelong treatment, causal gene must be identified, appropriate target tissue must be used

Question 30

what diseases have been treat with gene therapy?

Answer 30

SCID and hypercholesterolemia, certain froms of cancer