DNA techniques

Question 1

which scientific/technological techniques aided recombinant DNA technology?

Answer 1

discovery of structure of DNA
restriction nucleases
DNA ligation
DNA cloning
PCR
DNA sequencing

Question 2

what do restriction nucleases do?

Answer 2

allows DNA to be cleaved at specific sites, facilitating gene isolation and manipulation

Question 3

what does DNA ligation do?

Answer 3

makes it possible to design and construct DNA molecules not found in nature

Question 4

What does PCR do?

Answer 4

enables amplification of DNA

Question 5

what does DNA sequencing do?

Answer 5

rapid determination of sequence of nucleotides or whole genomes

Question 6

what is DNA ligase?

Answer 6

a specific type of enzyme that facilitates the joining of DNA strands together by catalysing the formation of a phosphodiester bonding

Question 7

what does DNA ligase play a role in?

Answer 7

repairing single-stranded breaks in duplex DNA in living organisms
and may also repair double-strand breaks

Question 8

what are the plasmid vectors most widely used for cloning?

Answer 8

small circular molecules of double stranded DNA derived from larger plasmids that occur naturally in bacterial cells

Question 9

how can we separate plasmid vectors from host DNA in cloning? how easy is this?

Answer 9

easy to separate
based on size using centrifugation

Question 10

how do restriction nucleases work?

Answer 10

purified plasmid vectors are cut with a restriction nuclease to create linear DNA molecules
DNA of interest is cut with same nucleases and resulting fragments are ligated together to form recombinant DNA circles using DNA ligase

Question 11

what was ‘baby blue’?

Answer 11

a prototype machine for doing PCR

Question 12

what did the discovery of PCR rely on?

Answer 12

use of Taq polymerase

Question 13

what are the steps of a polymerase chain reaction?

Answer 13

double stranded DNA is heated to separate strands
primers are hybridised
DNA polymerase is added
DNA synthesis from primers

Question 14

what does PCR stand for?

Answer 14

polymerase chain reaction

Question 15

what is Taq polymerase?

Answer 15

a DNA polymerase purified from T.aquaticus

Question 16

what are the properties of the DNA polymerase isolated from T.aquaticus?

Answer 16

stable at high temperatures

Question 17

how can genes be selectively amplified?

Answer 17

by PCR

Question 18

what are primers?

Answer 18

short sequences of DNA

Question 19

how are primers synthesised?

Answer 19

software

Question 20

what happens to the products of PCR?

Answer 20

run on agarose gels in an electric field and separated by size

Question 21

how is DNA synthesis on single strands started?

Answer 21

primed and begun using Taq polymerase

Question 22

What is DNA sequencing based on?

Answer 22

DNA polymerase

Question 23

how does DNA sequencing work?

Answer 23

four different chain terminating bases are used in four separate reactions, along with normal bases
each reaction produces a different set of products that terminate at different points in the sequence
products are separated by electrophoresis in PAGE
by reading off the bands in order, the sequence of DNA can be determined

Question 24

what is PAGE?

Answer 24

polyacrylamide gel

Question 25

what are the chain terminating bases used in DNA sequencing?

Answer 25

ddATP ddCTP ddGTP ddTTP

Question 26

what is the order in DNA sequencing (ie when reading off the bands)?

Answer 26

starting at bottom and working upwards

Question 27

how is DNA sequencing done now?

Answer 27

using technology

Question 28

what is reverse transcriptase?

Answer 28

an enzyme

Question 29

how can we distinguish between mRNA and tRNA/rRNA?

Answer 29

using a polyA tail

Question 30

how can we make cDNA clones?

Answer 30

short oligo complementary to 3' end of mRNA is hybridised to RNA to act as a primer for reverse transcriptase which copies RNA into complementary DNA DNA/RNA hybrid is treated with RNAase which creates gaps in RNA strand DNA polymerase copies single-stranded cDNA into double stranded cDNA molecules are inserted into virus or plasmid vectors and cloned

Question 31

what can be used to detect the presence of a viral genome in a nasal sample?

Answer 31

PCR

Question 32

what is an example of using PCR to detect viruses?

Answer 32

SARS-CoV-2 (genome is a single-strand molecule of RNA)

Question 33

how does illumina sequencing work?

Answer 33

a genome or other large DNA sample is broken into millions of short fragments these are attached to a glass surface and amplified by PCR to generate DNA clusters covering entire genome by taking snapshots of the glass slide as reactions progress, the sequence of nucleotides can be determined at scale