molecular techniques

Question 1

What are restriction enzymes?

Answer 1

endonucleases made by bacteria that cut at specific DNA sequences

Question 2

What is gene cloning

Answer 2

isolate gene using restriction enzyme 
gel extract 
ligate into plasmid vector 
transform into bacteria 
identify and isolate

Question 3

How does DNA electrophoresis

Answer 3

DNA negatively charged moves towards positive electrode

movement dependent on size of fragment

Question 4

Explain PCR

Answer 4

heat 95 to denature
cool to 60 +/- 5 primers anneal
extension at 72 degrees

Question 5

Explain protein DNA gel electrophoresis

Answer 5

similar DNA electrophoresis
movement dependent on size and charge of protein
density gives indication of number of molecules

Question 6

what does sds -page stand for

Answer 6

sodium dodecyl sulphate polyacrylamide gel electrophoresis

Question 7

What does SDS do

Answer 7

make protein linear remove charge
makes protein negatively charged
neg charge proportional to size of protein
as a result movement of protein on gel dependent only on size influence of charge removed

Question 8

What dye is used in sds page

Answer 8

coomassie dye

Question 9

How do you do protein identification

Answer 9

digest protein with trypsin
perform mas spec
generate lots if peptide sizes
use database of predicted peptide sizes for know proteins to identify

Question 10

How can ab’s be used

Answer 10

target specific epitope/ antigen

Question 11

explain western blot

Answer 11

transfer to nitrocellulose gel
apply primary ab
apply secondary enzyme linked ab
immunoblot

Question 12

What does ELISA stand for

Answer 12

enzyme linked immunoabsorbent assay

Question 13

Explain process of DNA hybridisation?

Answer 13

denature dsDNA to ssDNA
add labelled probe complementary to sequence of interest
identify usually using radioactive film

Question 14

What is a southern blot

Answer 14

identify sequences of interest after gel electrophoresis
gel transfer to nitrocelluse/ nylon membrane
alkaline solution to denature dsDNA
add probe
detect

Question 15

what is a northern blot

Answer 15

identify rna using dna probe similar to southern blot

Question 16

Key features of probes

Answer 16

do not need 100% similarity to sequence to bind, more similarity = greater binding strength
do not need to completely align with sequence they are being used to distinguish
do not affect position of band that is detected

Question 17

Explain sanger sequencing

Answer 17

use ddNTP of A/T/G/C which stop dna elongation due to missing 3’oh group
use one ddNTP in each tube with the normal dNTPs extension will be terminated at different points
run all sample on gel
read sequence off the gel bottom up

Question 18

How can identify mutants with PCR

Answer 18

run differently

allele specific primers

Question 19

how can you identify mutants with southern blotting

Answer 19

use allele specific probes

Question 20

Explain the process of RT-PCR

Answer 20

reverse transcriptase pcr
mRNA present if gene expressed 
use primer with long poly a tail 
bind mRNA 
then add reverse transcriptase to make cDNA 
add nuclease breaks down rna 
ssdna produced 
carrier out pcr as normal to amplify dsDNA molecule

Question 21

What does microarray allow? what are the two types ?

Answer 21

genome wide analysis of thousands of genes
conditional gene expression
array comparative genome hybridisation

Question 22

Explain process of conditional gene expression

Answer 22

abnormal vs normal cell
rna to dna (tagged fluoresecently ) using reverse transciptase
hybridised to microarray
read gene expression

Question 23

explain the process of array comparative genome hybridisation

Answer 23

normal vs abnormal
dna extracted and tagged
mixed in equal quantities and hybridised to microarray
read fluorescence
work out ratios of different fluorescence
decrease–> deletion
increase–> duplication
only one fluorescent- only present in one cell not the other

Question 24

What is a microarray

Answer 24

microscope slide with specific dna sequences dotted on it on specific regions

Question 25

Explain dna fingerprinting/ profiling

Answer 25

non coding regions contain short tandem repeats or mini satellites that are variable in an indivdual
re cut at specific gene loci and probe for mini staellites added
specific banding pattern seen on gel

Question 26

what is karotyping

Answer 26

look at chromosomes and banding pattern

Question 27

explain FISH

Answer 27

fluorescent in situ hybridisation 
DNA probe added to cell 
DNA denatured 
probe binds 
use control probe to find chromosome 
use mutant probe to identify mutant

Question 28

explain chromosome painting

Answer 28

probes made with specific fluorescent tag for each chromosome
each chromosome looks different colour
can identify mutation etc when colours mixed not just on one chromosome