Molecular Cloning And Gene Manipulation

Question 0

Restriction enzymes

Answer 0

Blunt end
5’ overhang
3’ overhang

Question 1

Genetic library prep

Genomic DNA from tissue

Answer 1

Digest so fragment fits in vector
For BAC
Ligation of inserts to prepare vector DNA

Question 2

What do plasmid vectors contain

Answer 2

Ori
Antibiotic resistance gene
Restriction sites

Question 3

Genomic library contains

Answer 3

Plasmid library
Phage library
A library of bacterial artificial chromosomes BAC clones

Question 4

DNA secuencing

Answer 4

Denature ddDNA
Add primer and DA(tagc)P 
Aliquot in 4 test tubes
Add appropriate dideoxy NTP and DNA polymerase
Run in matrix

Question 5

Methods of labelling

Answer 5

Original- radioactively label primer

Common- Fluorescent dideoxy

Question 6

What does ORF VERIFY

Answer 6

Gene family- southern blot
Expression-Northern blot
Transcription initiation site- DNA footprint and EMSA
EXON site - s1 nuclease mapping assay

Question 7

How does agarose separate DNA or RNA

Answer 7

Charge to mass ratio

Question 8

Newly synthesised DNA is either

Answer 8

Radioactively labeled (32p)
Or
Chemically DIG MODIFIED DTTP

Question 9

Southern blotting

Answer 9

Completely digest using several REnzymes
Label DNA probe with 32p DCTP
Allow binding the wash

Question 10

Using labelled DNA probes- hybridisation what happens

Answer 10

Ss DNA unbound washed away

Only dd radioactive DNA probes left and are visible on xray

Question 11

cDNA how does it work

Answer 11

Isolate mRNA by oligo(dt) column
Produce cDNA using oligo(dt) primer and reverse transcriptase
DNA polymerase produces second strand
DdDNA lighted with linkers with a endonuclease restriction site

Question 12

DNA foot printing what happens

Answer 12

End label with 32p
Allow TF to bind
Cleave at random
Denature DNA, separate by size
Xray

Question 13

ESMA How does it work

Answer 13

Prepare radioactively labeled DNA frag containing TF binding site
Incubate with tissue extract
Run on polyacrylamide gel
X-ray
Retarded bands indicate protein binding to DNA

Question 14

EMSA what does it do

Answer 14

Determine if the protein is capable of binding to a given DNA or RNA sequence

Question 15

S1 nuclease mapping

Answer 15

Label DNA with 32p
Denature DNA and hybridise with mRNA
Digest ssnucleic acid by s1 nuclease 
Run on polyacrylamide gel
X ray

Question 16

Northern blot

Answer 16

Same as southern

RNA separated by size

Question 17

Microarray what happens

Answer 17

Prepare a cDNA library and make a systemic assaying a solid surface representing 1 gene
Extract RNA and convert to cDNA 
Label cDNA with fluorescent dye cy3
Hybridise
Genes expressed are yellow cuz of dye

Question 18

Rt pcr

Answer 18

Reverse transcribe PCR
mrna prepared then converted to cDNA
Then gel electrophoresis shortest DNA travels furthest

Question 19

Qrt PCR WHAT MAKES IT DIFFERENT TO PCR

Answer 19

Uses fluorescent dyes
Allows to monitor amplification. In real time
Amplification measured exponentially

Question 20

Western blot SDS PAGE

Answer 20

Proteins
Denatured by negative SDS CHARGE
Negative charge allows proteins to migrate
Speed = net charge to mass ratio

Question 21

Gene approach to loss of function

Answer 21

Gene knock out

Question 22

Gene approach to gain of function

Answer 22

Gene knock in
Dominant negative analysis
Over expression

Question 23

Loss of function miss expression

Answer 23

mRNA injected into egg
At site different to normal
Detected with antisense probe labeled with DIG

Question 24

Gene knock out

Answer 24

Wt 4 petals
Mutation results in flours having lots
This indicated te AGAMOUS TF is regulating petal formation

Question 25

Gain of function
Dominant negative (dn) approach

Answer 25

Dn competes with wt

Dominant suppresses wt gene

Question 26

Dominant negative (dn) approach

Answer 26

Heterotrimeric g protein Galpha binds GTP
Galpha hydrolysed is inactive now
Inability to break down GTP
Constantly activated