DNA technology - gene cloning (1)

Question 1

stages

Answer 1

isolated gene
insert into vector - recombinant DNA
gel electrophoresis
DNA ligase join DNA fragment ligation

Question 2

gel electrophoresis

Answer 2

DNA + restriction E
along agarose gel and use UV light
control - remain single band at the top

Question 3

DNA ligase join DNA fragment ligation

Answer 3

catalyse phosphodiester bond - combination of DNA

put together plasmid and gene using ligase + ATP = recombination DNA

Question 4

type of ligase used mainly

Answer 4

T4 DNA ligase

Question 5

blunt end ligation

Answer 5

blunt ends join require more time to form bond

as it takes more time to form phosphodiester bonds

Question 6

sticky end ligation

Answer 6

overhangs form H bonds and stick and allow DNA ligase form bonds - more time given

Question 7

5 options for constructing recombinant DNA molecules

Answer 7

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

Question 8

cut vector and gene with same restriction

Answer 8

stick and bind

e.g. cut by BamH1

Question 9

vector and gene has different restriction E

Answer 9

BamH1 and Sal1

cuts them = sticky end and then bind together

Question 10

using nuclease and polymerase

Answer 10

nuclease = blunt ends e.g. Mng Bean nuclease
sticky ends not complementary - nuclease trims = blunt end
DNA polymerase = blunt ends - fill the overhang

Question 11

linker and adaptors

Answer 11

manipulate end of gene sequence

Question 12

linkers

Answer 12

sticks short pieces of DNA on gene = blunt

contains restriction E - BamH1 trim = sticky ends to plasmid

Question 13

adaptor

Answer 13

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

Question 14

TA cloning

Answer 14

manipulate to have specific nucleotide (A) at end gene and plasmid vector using terminal transferase
add complementary nucleotide (T)
increase chance DNA stick together

Question 15

transformation

Answer 15

intro of DNA into living cells

use heat and chemical = modify cell phenotype - take up DNA

Question 16

transposition into host cell after isolating DNA and insert into vector step

Answer 16

prepare competent bacterial cells
rapid heat up
selection

Question 17

prepare competent bacterial cells

Answer 17

treat normal bacterial cell with CaCl2 = changes charge of outer membrane - -ve DNA attracted

Question 18

rapid heat up

Answer 18

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

Question 19

selection

Answer 19

most vectors confer antibiotic resistance help determine which cells harbour recombinant DNA molecule
colonies transfer to agar + ampicillin plate

Question 20

recombinant vector

Answer 20

cute gene + cut vector + ligase + ATP

can also get self ligation (no gene)

Question 21

distinguishing bacteria between recombinant DNA/ self ligated vector

Answer 21

replica plating
blue/white
restriction mapping

Question 22

replica plating - pBR322 vectors

Answer 22

tetracyclin-selectable marker as recognition inactivation - does not function unlike self ligase

Question 23

replica plating - after transformation

Answer 23

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

Question 24

blue/white (pvc vector)

Answer 24

amp and Lac2 gene - selectable marker
self ligated produce galactosidase = blue
placed in amp + X-Gal - gene of interest remains white

Question 25

restriction mapping (any vector)

Answer 25

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

Question 26

expression vector

Answer 26

transcription | translation

Question 27

transcription

Answer 27

gene has bacterial promoter for transcription | RNA polymerase bind at 5' region - other pro and eukaryotic promoters are different

Question 28

why prokaryotic and eukaryotic promoters are different

Answer 28

prokaryotic contain prokaryotic sequence - lac promoter as well

Question 29

bacterial promoter regions

Answer 29

coding - centre 5' promoter - left 3' control - right

Question 30

translation of expression vector

Answer 30

make protein with right sequence - with promoter region and rbs

Question 31

rbs

Answer 31

ribosomal binding site | allow bacterial ribosomes engage with RNA = protein

Question 32

bacteria host cells characteristics

Answer 32

grown in light broth fast division doesn't glycosylate - no post-translational modification

Question 33

animal host cells characteristics

Answer 33

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

Question 34

example of animal host cell - factor VIII

Answer 34

for blood clotting - for haemophilia - synthesise factor VIII - purified from human blood

Question 35

example of animal host cell - production of recombinant factor VIIII

Answer 35

v. large - >186kb and 17 disulphide bridge use neonatal hamster cell - active after glycosylation use expression vector with SV40 promoter

Question 36

GM animal uses

Answer 36

pharmaceutical, vaccines and antibodies production source of cells, tissues and organs with no rejection produce high value of material, specific antimicrobial and efficient foor

Question 37

pharming

Answer 37

animals as bioreactor | gene of interest injected into using transgenic technology

Question 38

example of pharming - sheep

Answer 38

produce B-lactoglobulin milk

Question 39

example of pharming - goat

Answer 39

B-casein promoter

Question 40

example of pharming - chicken

Answer 40

lysozyme promoter

Question 41

value materials

Answer 41

surgical | all use same recombinant DNA technology

Question 42

human a antitrypsin (AAT)

Answer 42

inflammatory response - tissue distraction

Question 43

ATIII

Answer 43

anti thrombin III - prevent blood clotting

Question 44

gene therapy

Answer 44

insert pure copy of gene - treating disorder directly

Question 45

example of gene therapy - T cell

Answer 45

car T cell | T cell taken and replaced with modified to recognise cancer cells

Question 46

GM animal example - goat

Answer 46

produce spider silk in milk - fibres