enzyme and restriction mapping

Question 1

what are the three recombinant proteins

Answer 1

insulin
interferon - antiviral defence
G-CSF - promotes formation of bone marrow

Question 2

uses of transgenic organisms

Answer 2

disease models

improved agriculture yields

Question 3

what are nucleases

Answer 3

proteins that degrade nucleic acids by hydrolysing phosphodiester bonds

Question 4

what does RNase do

Answer 4

degrade RNA

Question 5

what does DNase do

Answer 5

degrade DNA

Question 6

what do exonuclease do?

Answer 6

degrade from the end of a molecule

Question 7

what do endonuclease do?

Answer 7

cleave within nucleotide chain

Question 8

what do restriction endonuclease do?

Answer 8

limit the transfer of nucleic acids from infecting phages into bacteria

Question 9

what do enzymes from restriction endonuclease do?

Answer 9

recognise specific sequences and cut that sequence

Question 10

what do restriction enzymes do (2)

Answer 10

recognise specific DNA sequences

catalyse the hydrolysis of phosphodiester bonds

Question 11

how often does a 4 base recognition sequence occur

Answer 11

every 256 bases

Question 12

how often does a 6 base recognition sequence occur

Answer 12

every 4096 bases

Question 13

when can nucleases produce

Answer 13

an overhang

a blunt end

Question 14

what are restriction enzymes crucial for

Answer 14

cloning
molecular diagnostics
characterisation of plasmids

Question 15

what DNA molecules an be joined together

Answer 15

human DNA e.g. insulin can be put with bacterial DNA e.g. plasmid

Question 16

what is sickle cell anaemia

Answer 16

mutation in beta globin gene

Question 17

what are restriction maps

Answer 17

map of restriction sites within a molecule
mapping on an unknown molecule
digest plasmid to give you fragment

Question 18

what happens when digested with bam HI

Answer 18

2 fragment results =
7kb
6kb

Question 19

what happens with Eco RI

Answer 19

1 linear fragment =

Question 20

what happens with a double digestion

Answer 20

results in 3 fragments
6kb
4kb
3kb

Question 21

what does DNA ligase do

Answer 21

repairs nicks In phosphodiester backbone

creates new phosphodiester bonds

Question 22

why use a DNA polymerase

Answer 22

PCR amplification
generation of probes
blunt-ending of DNA overhangs

Question 23

what does DNA polymerase do

Answer 23

DNA synthesis 5’-3’

extends DNA fragment

Question 24

function of phosphatase

Answer 24

hydrolyses a phosphate group of its substrate

Question 25

where are two places you can get phosphatase

Answer 25

calf intestinal alkaline phosphatase

shrimp alkaline phosphatase

Question 26

why use a phosphatase

Answer 26

to prevent cut plasmids from resealing

Question 27

describe polynucleotide kinase

Answer 27

kinase: phosphate from ATP to substrate

polynucleotide kinase adds phosphate to 5’ hydroxyl group of DNA or RNA

Question 28

why use a polynucleotide kinase

Answer 28

• phosphorylate chemically snythesized DNA so can ligate to other fragment
  label dna to be traced radioactively or fluorescently

Question 29

describe probes

Answer 29

fragments of single stranded DNA
20-1000 bases
complementary to gene of interest

Question 30

describe reverse transcriptase

Answer 30

RNA dependent DNA polymerase
isolated from RNA containing retroviruses
synthesises a dna molecule complementary to a mRNA template using dNTPs

Question 31

describe random primers

Answer 31

cDNAs up to 700bp but cover all length of the RNA molecules

Question 32

describe oligo (dT) primers

Answer 32

useful for cloning cDNAs and cDNA libraries, some may be full length