DNA technology - gene cloning

Question 1

benefits

Answer 1

small nucleotides sequences/genes - medical

everyday life - pest-resistant crops - bacteria cloned to rid toxins

Question 2

1st - 1900s mendalian

Answer 2

theory hereditary

inheritable protein depend on factor

Question 3

2nd - 1950-60s

Answer 3

generic code cracked - generic material, transcription and translation

Question 4

3rd - 1970

Answer 4

recombinant DNA - cut and reassemble pieces of DNA

genome - length of DNA in one haploid set of chromosome - entire sets

Question 5

gene cloning

Answer 5

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

Question 6

importance of DNA technology

Answer 6

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

Question 7

gene sequence and expression

Answer 7

molecular bio gene function and regulation
understand sequence and how it is expressed
know the mutagenesis experiment - medical

Question 8

detect mutation

Answer 8

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

Question 9

develop new ways to treat disease

Answer 9

diabetes, insulin

Question 10

cure inherited diseases

Answer 10

using gene therapy

e.g. CFTR

Question 11

gene cloning - isolating gene

Answer 11

using PCR or southern blotting and hybridisation

Question 12

what PCR requires

Answer 12

require DNA template, 2 oligonucleotide primers, polymerase and nucleotide

Question 13

PCR processes - needed, repeated

Answer 13

denature
annealing
extension
repeat 35 times but >35 = +ve effect - reagent - depleted and DNA polymerase is damaged

Question 14

Denature

Answer 14

at 95 degrees

denature template DsDNA = ssDNA

Question 15

annealing

Answer 15

at 55 degrees

primer anneal to complementary DsDNA

Question 16

extension

Answer 16

at 72 degrees

DNA polymerase extends primer opt temp

Question 17

discovering polymerase in bacteria - thermus aquaticus

Answer 17

opt 72 degrees but heat stable to 94 degrees

Question 18

checking PCR - on agarose gel

Answer 18

amplify region - one distinct bond
generate enough DNA - sequencing and detect mutation
enough DNA digest - clone into expression = protein

Question 19

limitation

Answer 19

difficult as amplify product longer than 10-15kb

need to know sequence flanking your gene

Question 20

southern blotting and hybridisation

Answer 20

cut human DNA into fragment using ECORI - restriction E

run on agarose gel = laddering

Question 21

southern blotting (1)

Answer 21

transfer DNA fragment onto nitrocellulose filter and hybridisation - identify band of interest

Question 22

southern blotting - genome + radiolabelled isotope 32P

Answer 22

small fraction of sequence - code for gene make primer - heat = single probes and hybrids with filter

Question 23

southern blotting - using X-ray film

Answer 23

rerun gel and compare

X-ray shows gel of interest - cut out band and sequence cloned

Question 24

insertion in vector = recombinant DNA

Answer 24

plasmid vector

restriction E

Question 25

plasmid vector

Answer 25

produce large number of DNA derived by plasmid

multiply using host cell machinery and cell divides

Question 26

plasmid vector - origin of replication

Answer 26

vector has site to replicate - 100-1000

Question 27

example plasmid vector - pBR322

Answer 27

4362 bp

<10000bp easily puried avoid DNA breakdown - smaller size plasmid = more insert

Question 28

bacteria without vector

Answer 28

vector contains antibiotic resistance gene

and when removed - bacteria dies in culture

Question 29

restriction E function

Answer 29

sites in plasmids to cut open

Question 30

Restriction E in bacteria

Answer 30

protect from bacteriophage and infection(virus)

Question 31

restriction E mechanism (cut DsDNA)

Answer 31

cut DsDNA cut at specific nucleotide sequence by hydrolysis of phosphodiester bonds

Question 32

restriction E mechanism (ECORI)

Answer 32

enzyme CORI-1 (Ecoli) binds to GAATTC in DNA and cut
cannot cut own DNA
contains ECOR1 methylase - methylates GAATTC - prevent binding

Question 33

DNA methylation

Answer 33

as it is semi-conservative replication - one strand is methylated
DNA methylase - methylates hemi- methylate DNA

Question 34

hemi methylate DNA

Answer 34

add methyl group at the strand with a template that has already been methylated

Question 35

types of restriction E

Answer 35

exonuclease

endonuclease

Question 36

exonuclease

Answer 36

remove nucleotide from ends

Question 37

endonuclease

Answer 37

break nucleic acid chain in sequence

3 types

Question 38

endonuclease type 1

Answer 38

cut at 1000 base from RS

Question 39

endonuclease type 2

Answer 39

cut at RS

e.g. ECOR1

Question 40

endonuclease type 3

Answer 40

cut 25 bases from RS

Question 41

RS

Answer 41

recognition site
many recognise hexanucleotide
4,5,8 nucleotide site

Question 42

naming e.g. ECOR1

Answer 42

E - genus
CO - species of prokaryotic cell
number - order E - isolated from single strain bacteria

Question 43

types of cuts

Answer 43

blunt end cutter

sticky end cutter

Question 44

blunt end cutter

Answer 44

attach to specific binding site and cut straight down DsDNA

Question 45

sticky end cutter

Answer 45

bind to specific sequence and cause staggered cut - overcut = ss sequence bind to other complementary strands easily bind to plasmid