enzyme and restriction mapping Flashcards
what are the three recombinant proteins
insulin
interferon - antiviral defence
G-CSF - promotes formation of bone marrow
uses of transgenic organisms
disease models
improved agriculture yields
what are nucleases
proteins that degrade nucleic acids by hydrolysing phosphodiester bonds
what does RNase do
degrade RNA
what does DNase do
degrade DNA
what do exonuclease do?
degrade from the end of a molecule
what do endonuclease do?
cleave within nucleotide chain
what do restriction endonuclease do?
limit the transfer of nucleic acids from infecting phages into bacteria
what do enzymes from restriction endonuclease do?
recognise specific sequences and cut that sequence
what do restriction enzymes do (2)
recognise specific DNA sequences
catalyse the hydrolysis of phosphodiester bonds
how often does a 4 base recognition sequence occur
every 256 bases
how often does a 6 base recognition sequence occur
every 4096 bases
when can nucleases produce
an overhang
a blunt end
what are restriction enzymes crucial for
cloning
molecular diagnostics
characterisation of plasmids
what DNA molecules an be joined together
human DNA e.g. insulin can be put with bacterial DNA e.g. plasmid
what is sickle cell anaemia
mutation in beta globin gene
what are restriction maps
map of restriction sites within a molecule
mapping on an unknown molecule
digest plasmid to give you fragment
what happens when digested with bam HI
2 fragment results =
7kb
6kb
what happens with Eco RI
1 linear fragment =
what happens with a double digestion
results in 3 fragments
6kb
4kb
3kb
what does DNA ligase do
repairs nicks In phosphodiester backbone
creates new phosphodiester bonds
why use a DNA polymerase
PCR amplification
generation of probes
blunt-ending of DNA overhangs
what does DNA polymerase do
DNA synthesis 5’-3’
extends DNA fragment
function of phosphatase
hydrolyses a phosphate group of its substrate
where are two places you can get phosphatase
calf intestinal alkaline phosphatase
shrimp alkaline phosphatase
why use a phosphatase
to prevent cut plasmids from resealing
describe polynucleotide kinase
kinase: phosphate from ATP to substrate
polynucleotide kinase adds phosphate to 5’ hydroxyl group of DNA or RNA
why use a polynucleotide kinase
- phosphorylate chemically snythesized DNA so can ligate to other fragment
label dna to be traced radioactively or fluorescently
describe probes
fragments of single stranded DNA
20-1000 bases
complementary to gene of interest
describe reverse transcriptase
RNA dependent DNA polymerase
isolated from RNA containing retroviruses
synthesises a dna molecule complementary to a mRNA template using dNTPs
describe random primers
cDNAs up to 700bp but cover all length of the RNA molecules
describe oligo (dT) primers
useful for cloning cDNAs and cDNA libraries, some may be full length
