DNA technology - gene cloning Flashcards
benefits
small nucleotides sequences/genes - medical
everyday life - pest-resistant crops - bacteria cloned to rid toxins
1st - 1900s mendalian
theory hereditary
inheritable protein depend on factor
2nd - 1950-60s
generic code cracked - generic material, transcription and translation
3rd - 1970
recombinant DNA - cut and reassemble pieces of DNA
genome - length of DNA in one haploid set of chromosome - entire sets
gene cloning
1) gene isolated from particular tissue/cell type
2) insert DNA into cloning vector - recombinant DNA
3) introduce recombinant DNA to host cell - Ecoli bacteria
4) multiplication of recombinant DNA
importance of DNA technology
general large amount of DNA and protein - large region, pure sample of gene gene sequence and expression detect mutation develop new ways to treat disease cure inherited diseases
gene sequence and expression
molecular bio gene function and regulation
understand sequence and how it is expressed
know the mutagenesis experiment - medical
detect mutation
genetic diseases - neonatal/prenatal screening - for common genetic disorder - CF
presymptomatic testing in late on set genetic disease and Huntington’s disease e.g. familial colon cancer - inherited breast/ovation cancer
develop new ways to treat disease
diabetes, insulin
cure inherited diseases
using gene therapy
e.g. CFTR
gene cloning - isolating gene
using PCR or southern blotting and hybridisation
what PCR requires
require DNA template, 2 oligonucleotide primers, polymerase and nucleotide
PCR processes - needed, repeated
denature
annealing
extension
repeat 35 times but >35 = +ve effect - reagent - depleted and DNA polymerase is damaged
Denature
at 95 degrees
denature template DsDNA = ssDNA
annealing
at 55 degrees
primer anneal to complementary DsDNA
extension
at 72 degrees
DNA polymerase extends primer opt temp
discovering polymerase in bacteria - thermus aquaticus
opt 72 degrees but heat stable to 94 degrees
checking PCR - on agarose gel
amplify region - one distinct bond
generate enough DNA - sequencing and detect mutation
enough DNA digest - clone into expression = protein
limitation
difficult as amplify product longer than 10-15kb
need to know sequence flanking your gene
southern blotting and hybridisation
cut human DNA into fragment using ECORI - restriction E
run on agarose gel = laddering
southern blotting (1)
transfer DNA fragment onto nitrocellulose filter and hybridisation - identify band of interest
southern blotting - genome + radiolabelled isotope 32P
small fraction of sequence - code for gene make primer - heat = single probes and hybrids with filter
southern blotting - using X-ray film
rerun gel and compare
X-ray shows gel of interest - cut out band and sequence cloned
insertion in vector = recombinant DNA
plasmid vector
restriction E
plasmid vector
produce large number of DNA derived by plasmid
multiply using host cell machinery and cell divides
plasmid vector - origin of replication
vector has site to replicate - 100-1000
example plasmid vector - pBR322
4362 bp
<10000bp easily puried avoid DNA breakdown - smaller size plasmid = more insert
bacteria without vector
vector contains antibiotic resistance gene
and when removed - bacteria dies in culture
restriction E function
sites in plasmids to cut open
Restriction E in bacteria
protect from bacteriophage and infection(virus)
restriction E mechanism (cut DsDNA)
cut DsDNA cut at specific nucleotide sequence by hydrolysis of phosphodiester bonds
restriction E mechanism (ECORI)
enzyme CORI-1 (Ecoli) binds to GAATTC in DNA and cut
cannot cut own DNA
contains ECOR1 methylase - methylates GAATTC - prevent binding
DNA methylation
as it is semi-conservative replication - one strand is methylated
DNA methylase - methylates hemi- methylate DNA
hemi methylate DNA
add methyl group at the strand with a template that has already been methylated
types of restriction E
exonuclease
endonuclease
exonuclease
remove nucleotide from ends
endonuclease
break nucleic acid chain in sequence
3 types
endonuclease type 1
cut at 1000 base from RS
endonuclease type 2
cut at RS
e.g. ECOR1
endonuclease type 3
cut 25 bases from RS
RS
recognition site
many recognise hexanucleotide
4,5,8 nucleotide site
naming e.g. ECOR1
E - genus
CO - species of prokaryotic cell
number - order E - isolated from single strain bacteria
types of cuts
blunt end cutter
sticky end cutter
blunt end cutter
attach to specific binding site and cut straight down DsDNA
sticky end cutter
bind to specific sequence and cause staggered cut - overcut = ss sequence bind to other complementary strands easily bind to plasmid
