experimental techniques

Question 1

ELISA

Answer 1

detects either antigens or antibodies

1. sample added to wells, each well coated with Ab specific for the antigen
2. secondary Ab with enzyme-link added
3. coloured substrate shows you where it is

Question 2

Radioimmunoassay

Answer 2

similar to ELISAs but use radiolabled antigen and antibodies rather than an enzyme-link

1. known amount of radiolabeled antigen added.
2. known amount of Ab added; radiation measured
3. sample is added in increasing amount, the new antigen ought to displace radiolabeled antigen, so you measure radioactivity change

Question 3

blotting

Answer 3

southern - DNA
Northern - RNA
Western - proteins
after gel electrophoresis, proteins/DNA transferredto nitrocellulose membrane, detected via radiolabeled probe (which may be complementary DNA, or Ab for the protein)

Question 4

DNA recombination

Answer 4

a gene for a certain protein can be placed in a bacterial plasmid using restriction enzymes. The bacteria take up the plasmid, can be ‘farmed’ for the protein.

Question 5

bacterial transformation

Answer 5

Early experiment with rough and smooth strain.

Bacteria must be heat shocked to facilitate plasmid uptake. The plasmid may or may not be taken up into genome too

Question 6

artificial chromosomes

Answer 6

plasmids only used for small inserts. Large inserts use artificial chromosomes!!

Question 7

eukaryotic plasmids

Answer 7

much the same as bacterial plasmids, but have polyadenylation signal downstream of gene to terminate transcription.
Introduced to cells via TRANSFECTION (method without viruses) or TRANSDUCTION (viruses use reverse transcriptase to put gene in your genome)

Question 8

2 types of PCR

Answer 8

RT-PCR is good for detecting mRNAs

qPCR (q for quantitative) detects amounts. Uses a fluorescent dye that binds DNA

Question 9

Sanger sequencing

Answer 9

terminates translation NT by NT by using ddNTPs instead of usual dNTP susbrate (the ddNTPs lack the hydroxyl on 3’ so nothing can be added after

Question 10

DNA fingerprinting

Answer 10

uses DNA POLYMORPHISMS, repeating stretches of noncoding DNA

1. restriction fragment length polymorphism - these are digested and electrophoresed
2. short tandem repeat analysis - you amplify using PCR and then electrophorese

Question 11

exome and targeted sequencing

Answer 11

sequence only the exomes

Question 12

karyotyping

Answer 12

stain and visualize chromosomes

Question 13

fluorescence in situ hybridization

Answer 13

use fluorescent probes to locate specific DNA sequence on chromosomes (visualize where they are)

Question 14

microarrays

Answer 14

study relative RNA levels (microarray wells will show up red, green, or yellow)

Question 15

in situ hybridization

Answer 15

very thin slice of tissue is permeablizied to open cell membrane, and a labeled probe is added to bind to mRNA

Question 16

immunohistochemistry

Answer 16

antigen is recognized by Ab, which is recognized by secondary Ab, which is linked to an enzyme or fluorescent molecule

Question 17

flow cytometry

Answer 17

single cells are stained for certain protein markers using specific antibodies (with fluorescent tag). The fluorescence amount will then reveal how much protein there is

Question 18

siRNA

Answer 18

can bind to mRNA and decrease activity or promote their degradation