DNA technology - gene cloning (1) Flashcards
stages
isolated gene
insert into vector - recombinant DNA
gel electrophoresis
DNA ligase join DNA fragment ligation
gel electrophoresis
DNA + restriction E
along agarose gel and use UV light
control - remain single band at the top
DNA ligase join DNA fragment ligation
catalyse phosphodiester bond - combination of DNA
put together plasmid and gene using ligase + ATP = recombination DNA
type of ligase used mainly
T4 DNA ligase
blunt end ligation
blunt ends join require more time to form bond
as it takes more time to form phosphodiester bonds
sticky end ligation
overhangs form H bonds and stick and allow DNA ligase form bonds - more time given
5 options for constructing recombinant DNA molecules
cute vector and gene with same restriction E
vector and gene has different restriction E
using nuclease and polymerase
using linker and adapters
TA cloning
cut vector and gene with same restriction
stick and bind
e.g. cut by BamH1
vector and gene has different restriction E
BamH1 and Sal1
cuts them = sticky end and then bind together
using nuclease and polymerase
nuclease = blunt ends e.g. Mng Bean nuclease
sticky ends not complementary - nuclease trims = blunt end
DNA polymerase = blunt ends - fill the overhang
linker and adaptors
manipulate end of gene sequence
linkers
sticks short pieces of DNA on gene = blunt
contains restriction E - BamH1 trim = sticky ends to plasmid
adaptor
overhanging sticky ends bind to gene
all overhang for this has back phosphate group - no phosphodiester bonds until it binds to gene to gene and wash off other adaptor
use polynucleotide kinase to put back phosphate groups
TA cloning
manipulate to have specific nucleotide (A) at end gene and plasmid vector using terminal transferase
add complementary nucleotide (T)
increase chance DNA stick together
transformation
intro of DNA into living cells
use heat and chemical = modify cell phenotype - take up DNA
transposition into host cell after isolating DNA and insert into vector step
prepare competent bacterial cells
rapid heat up
selection
prepare competent bacterial cells
treat normal bacterial cell with CaCl2 = changes charge of outer membrane - -ve DNA attracted
rapid heat up
2mins at 42 degrees
heat shock = membrane fluid
phospholipid become more mobile - DNA pass easily
grown in agar plat - spread cells grow individual colonies
0.01% - plasmids taken up as inefficient process of transformation
selection
most vectors confer antibiotic resistance help determine which cells harbour recombinant DNA molecule
colonies transfer to agar + ampicillin plate
recombinant vector
cute gene + cut vector + ligase + ATP
can also get self ligation (no gene)
distinguishing bacteria between recombinant DNA/ self ligated vector
replica plating
blue/white
restriction mapping
replica plating - pBR322 vectors
tetracyclin-selectable marker as recognition inactivation - does not function unlike self ligase
replica plating - after transformation
placed in ampicillin plate then make replicate on agar + tet
bacteria + gene of interest dies
compare where gene of interest is located on the amp agar plate
blue/white (pvc vector)
amp and Lac2 gene - selectable marker
self ligated produce galactosidase = blue
placed in amp + X-Gal - gene of interest remains white
restriction mapping (any vector)
selection process - exterior DNA
using restriction E - recombinant or self ligated vector
hit piece of DNA - size of DNA fragments in agarose gel
self ligated - show single bond
recombinant - small bands - size of gene of interest
expression vector
transcription
translation
transcription
gene has bacterial promoter for transcription
RNA polymerase bind at 5’ region - other pro and eukaryotic promoters are different
why prokaryotic and eukaryotic promoters are different
prokaryotic contain prokaryotic sequence - lac promoter as well
bacterial promoter regions
coding - centre
5’ promoter - left
3’ control - right
translation of expression vector
make protein with right sequence - with promoter region and rbs
rbs
ribosomal binding site
allow bacterial ribosomes engage with RNA = protein
bacteria host cells characteristics
grown in light broth
fast division
doesn’t glycosylate - no post-translational modification
animal host cells characteristics
grown attached to solid matrix
divide every 18hrs
glycosylate protein - post-translational modification
uses animal expression vectors in monolayer culture in multiple vessels
e.g. liver/kidney cell ions
example of animal host cell - factor VIII
for blood clotting - for haemophilia - synthesise factor VIII - purified from human blood
example of animal host cell - production of recombinant factor VIIII
v. large - >186kb and 17 disulphide bridge
use neonatal hamster cell - active after glycosylation
use expression vector with SV40 promoter
GM animal uses
pharmaceutical, vaccines and antibodies production
source of cells, tissues and organs with no rejection
produce high value of material, specific antimicrobial and efficient foor
pharming
animals as bioreactor
gene of interest injected into using transgenic technology
example of pharming - sheep
produce B-lactoglobulin milk
example of pharming - goat
B-casein promoter
example of pharming - chicken
lysozyme promoter
value materials
surgical
all use same recombinant DNA technology
human a antitrypsin (AAT)
inflammatory response - tissue distraction
ATIII
anti thrombin III - prevent blood clotting
gene therapy
insert pure copy of gene - treating disorder directly
example of gene therapy - T cell
car T cell
T cell taken and replaced with modified to recognise cancer cells
GM animal example - goat
produce spider silk in milk - fibres
