lecture 5 - 08/10/24

Question 1

What is in vitro?

Answer 1

in the lab

Question 2

What is in vivo?

Answer 2

in organism

Question 3

ingredients for PCR

Answer 3

Template DNA
Primers
dNTPs
Buffer (Mg 2+)
taq polymerase

Question 4

What is taq polymerase?

Answer 4

modified DNA polymerase
from bacterial species that can survive hotter temperatures (originally in hot springs, Yellowstone)

Question 5

What are the 3 steps in PCR?

Answer 5

1. Denaturation
2. Annealing
3. Extension

Question 6

Denaturation

Answer 6

double DNA strand melts open

Question 7

Annealing

Answer 7

primers bind to DNA and polymerase attaches and starts copying DNA

Question 8

Extension

Answer 8

at 72oC: optimum temperature for taq polymerase and extension of fragment

Question 9

What is the optimum temperature of DNA polymerase?

Answer 9

37oC (body temp)

Question 10

Is PCR amplification linear or exponential?

Answer 10

exponential
e.g. 30 cycles = 20^30

Question 11

Which bits of the DNA is amplified?

Answer 11

only DNA between primers is amplified

ends of the amplified fragment are defined by 2 primers

(as only replicating a specific section, NOT replicating the genome)

Question 12

Primers in PCR

Answer 12

self designed to cater for the specific region needed
~20 bp oligonucleotides

Question 13

What is agarose?

Answer 13

complex polysaccharide

Question 14

separation of DNA by agarose gel electrophoresis

Answer 14

electric current is applied to gel
DNA moves to + electrode because it is - charged
moves through gel depending on:
- conformation (linear, circular, supercoiled)
- size

Question 15

How does size affect DNA movement in agarose gel electrophoresis?

Answer 15

smaller fragments move through the gel faster than large ones

Question 16

How to detect DNA in agarose gel electrophoresis?

Answer 16

stain DNA with fluorescent dye for detection by UV exposure
e.g. ethidium bromide - intercalates + binds w/ DNA

Question 17

What is the ladder used for in agarose gel electrophoresis?

Answer 17

used to identify each band size
- standardises sizes

Question 18

Uses of PCR

Answer 18

detection of pathogens in water
DNA sequencing
Diagnosis of genetic disorders
Prenatal diagnosis
Analysis of ancient DNA
Genetic fingerprinting
Forensic analysis

Question 19

Limitations of PCR

Answer 19

1. sequence information is required to design 2
   primers
2. limit on length of amplified fragment
3. potentially high error rate e.g. Taq polymerase =
   ~10^-4
4. very sensitive to exact reaction conditions, not
   easily quantified
5. tiny amounts of contaminating DNA will also be
   amplified

Question 20

Sanger Sequencing ‘ingredients’

Answer 20

DNA polymerase
dNTPs +ddATP, ddTTP, ddGTP, ddCTP
template DNA
Primer
DNA synthesis 5’ to 3’ direction

Question 21

What is a dNTP?

Answer 21

normal deoxyribonucleoside triphosphate

3’ OH allows strand extension at 3’ end

Question 22

What is a chain-terminating ddNTP?

Answer 22

chain-terminating dideoxyribonucleoside triphosphate

coloured

terminates the sequence as no 3’ -OH

Question 23

The principle of Sanger sequencing

Answer 23

1. add primer to single-stranded DNA fragment to
   be sequenced
2. add small amounts of labelled chain-terminating
   ddNTPs
3. add excess amounts of unlabelled dNTPs
4. mixture of DNA products (each containing a
   chain-terminating ddNTP labelled with a specific
   fluorescent marker
5. products loaded onto capillary gel
6. electrophoresis, size-separated products are
   read in sequence

Question 24

What shape chromosome are bacterial genomes?

Answer 24

circular

Question 25

Do all bacteria have plasmids are what are they?

Answer 25

not all but most
plasmids are small extrachromosomal circles of DNA

Question 26

What kind of enzyme cuts DNA?

Answer 26

restriction endonucleases
cuts nucleic acid sequences
sequence must be palindromic (5’ –> 3’ = 3’ –> 5’)

Question 27

How often might we expect this enzyme, HindIII, to cut the E. coli genome?

5’ –AAGCTT–3’

Answer 27

1/4 x 1/4 x 1/4 x 1/4 x 1/4 x 1/4 = 1/4096

E coli genome ~ 4.6Mb long

(4.5x10^6) / 4096
= 1123 sites

Question 28

What does DNA ligase do?

Answer 28

forms phosphodiester bonds
requires ATP

Question 29

Describe gene cloning

Answer 29

1. introduce recombinant plasmid into bacterial cell
2. will be replicated
3. cell will divide
4. clone of cells
5. recover DNA for analysis
6. gene cloning

(can also clone genes by insertion into a bacteriophage DNA vectors)

Question 30

What does it mean by the genetic code is universal?

Answer 30

it is the same in all organisms

Question 31

Transgenics

Answer 31

• genes between species
• genetic code is universal
• it is possible to take a gene from one organism
  and express it in another

e.g. production of insulin