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BIOL1024 Fundamentals of Biochemistry > DNA technology - gene cloning (1) > Flashcards

DNA technology - gene cloning (1) Flashcards

1
Q

stages

A

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

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2
Q

gel electrophoresis

A

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

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3
Q

DNA ligase join DNA fragment ligation

A

catalyse phosphodiester bond - combination of DNA

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

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4
Q

type of ligase used mainly

A

T4 DNA ligase

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5
Q

blunt end ligation

A

blunt ends join require more time to form bond

as it takes more time to form phosphodiester bonds

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6
Q

sticky end ligation

A

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

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7
Q

5 options for constructing recombinant DNA molecules

A

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

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8
Q

cut vector and gene with same restriction

A

stick and bind

e.g. cut by BamH1

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9
Q

vector and gene has different restriction E

A

BamH1 and Sal1

cuts them = sticky end and then bind together

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10
Q

using nuclease and polymerase

A

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

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11
Q

linker and adaptors

A

manipulate end of gene sequence

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12
Q

linkers

A

sticks short pieces of DNA on gene = blunt

contains restriction E - BamH1 trim = sticky ends to plasmid

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13
Q

adaptor

A

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

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14
Q

TA cloning

A

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

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15
Q

transformation

A

intro of DNA into living cells

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

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16
Q

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

A

prepare competent bacterial cells
rapid heat up
selection

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17
Q

prepare competent bacterial cells

A

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

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18
Q

rapid heat up

A

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

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19
Q

selection

A

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

20
Q

recombinant vector

A

cute gene + cut vector + ligase + ATP

can also get self ligation (no gene)

21
Q

distinguishing bacteria between recombinant DNA/ self ligated vector

A

replica plating
blue/white
restriction mapping

22
Q

replica plating - pBR322 vectors

A

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

23
Q

replica plating - after transformation

A

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

24
Q

blue/white (pvc vector)

A

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

25
Q

restriction mapping (any vector)

A

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

26
Q

expression vector

A

transcription

translation

27
Q

transcription

A

gene has bacterial promoter for transcription

RNA polymerase bind at 5’ region - other pro and eukaryotic promoters are different

28
Q

why prokaryotic and eukaryotic promoters are different

A

prokaryotic contain prokaryotic sequence - lac promoter as well

29
Q

bacterial promoter regions

A

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

30
Q

translation of expression vector

A

make protein with right sequence - with promoter region and rbs

31
Q

rbs

A

ribosomal binding site

allow bacterial ribosomes engage with RNA = protein

32
Q

bacteria host cells characteristics

A

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

33
Q

animal host cells characteristics

A

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

34
Q

example of animal host cell - factor VIII

A

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

35
Q

example of animal host cell - production of recombinant factor VIIII

A

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

36
Q

GM animal uses

A

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

37
Q

pharming

A

animals as bioreactor

gene of interest injected into using transgenic technology

38
Q

example of pharming - sheep

A

produce B-lactoglobulin milk

39
Q

example of pharming - goat

A

B-casein promoter

40
Q

example of pharming - chicken

A

lysozyme promoter

41
Q

value materials

A

surgical

all use same recombinant DNA technology

42
Q

human a antitrypsin (AAT)

A

inflammatory response - tissue distraction

43
Q

ATIII

A

anti thrombin III - prevent blood clotting

44
Q

gene therapy

A

insert pure copy of gene - treating disorder directly

45
Q

example of gene therapy - T cell

A

car T cell

T cell taken and replaced with modified to recognise cancer cells

46
Q

GM animal example - goat

A

produce spider silk in milk - fibres

BIOL1024 Fundamentals of Biochemistry (25 decks)

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