Molecular Biology 3

Question 1

what are mutations?

Answer 1

permanent alteration in a DNA (gene) sequence

Question 2

what causes mutations?

Answer 2

errors in DNA synthesis that can occur spontaneously at low frequency, chemical mutagens, ionising radiation

Question 3

what are the different types of substitution mutations?

Answer 3

conservative, non-conservative and no mutation

Question 4

what is a conservative mutation?

Answer 4

amino acid is replaced by one with similar properties

Question 5

what is a non-conservation mutation?

Answer 5

amino acid is replaced by one with different properties

Question 6

what is no mutations?

Answer 6

a change in the third position of the codon does not change an amino acid

Question 7

what is the effect of an insertion/deletion?

Answer 7

causes a frame-shift which synthesises a protein that has a completely different sequences downstream of the mutation

Question 8

what does a stop codon do?

Answer 8

premature or delayed termination

Question 9

what is sickle cell anaemia caused by?

Answer 9

single-base substitution in the beta-chain of haemoglobin

Question 10

what is the substitution in sickle cell anaemia?

Answer 10

glutamic acid is replaced with valine

Question 11

what does gene cloning produce?

Answer 11

large numbers of copies of a particular piece of DNA

Question 12

how are genes usually cloned?

Answer 12

by isolating them using restriction enzymes, gel electrophoresis and then inserting into a plasmid

Question 13

what do restriction enzymes do?

Answer 13

cut double-stranded DNA at specific DNA sequences

Question 14

what is good about sticky ends

Answer 14

they allow DNA fragments to re-associate by base pairing

Question 15

what is gel electrophoresis used to do?

Answer 15

separate DNA fragments on basis of their size

Question 16

what is step 1 of gene cloning?

Answer 16

restriction enzyme is chosen that cuts on either side of the gene but not in the middle

Question 17

what is step 2 of gene cloning

Answer 17

the gene is separated from other DNA fragments by gel electrophoresis

Question 18

what is step 3 of gene cloning

Answer 18

suitable plasmid is linearised (cut at one point) using the same restriction enzyme

Question 19

what is step 4 of gene cloning

Answer 19

the cut plasmid and gene are mixed, and the sticky ends of the plasmid and gene are allowed to anneal

Question 20

what is step 5 of gene cloning

Answer 20

the annealed ends are covalently joined using DNA ligase

Question 21

what is step 6 of gene cloning

Answer 21

the plasmid, now containing the gene of interest, is introduced into the host bacterium

Question 22

what is step 7 of gene cloning?

Answer 22

the bacteria are grown into a colony, using antibiotic resistance genes in the plasmid to select colonies containing plasmids

Question 23

what is step 8 of gene cloning

Answer 23

cloned cells are lysed and the plasmids isolated by centrifugation

Question 24

what is step 9 of gene cloning

Answer 24

plasmids are cut with the restriction enzyme, releasing the cloned gene

Question 25

what is DNA sequencing used for?

Answer 25

used to determine base sequences of DNA

Question 26

what does DNA sequencing do?

Answer 26

work out the structure of a gene or an entire genome

Question 27

what are the reaction components of DNA sequencing?

Answer 27

single-stranded DNA template, primer, deoxynucleotides, dideoxynucleotides, DNA polymerase, label

Question 28

what is step 1 of DNA sequencing

Answer 28

DNA to be sequenced is mixed with primer

Question 29

what is step 2 of DNA sequencing

Answer 29

primer binds to 3’ end of DNA

Question 30

what is step 3 of DNA sequencing

Answer 30

DNA primer mecture divided into 2 separate reaction tubes

Question 31

what is step 4 of DNA sequencing

Answer 31

chain synthesis proceeds in each of the four reaction mixtures

Question 32

what is step 5 in DNA sequencing

Answer 32

gel electrophoresis separation of reaction products - band corresponding to each position of chain termination appears

Question 33

what is step 6 of DNA sequencing

Answer 33

DNA bands detected by autoradiography or by laser in an automated sequencer

Question 34

what is step 7 of DNA sequencing

Answer 34

DNA sequence can be deduced from the pattern of bands in the four lanes