lecture 3

Question 1

DNA sequencing method using

Answer 1

Polymerase Chain Reaction (PCR)

Question 2

what does PCR

Answer 2

amplifies the DNA by repetitively cycles of denaturing and renaturing of DNA in the presence of thermostable DNA polymerase

need 2 primers that anneal to teh ends of amplified DNA fragment at 50 - 60 degreesC

Taq polymerase synthesizes new strands of DNA starting at the 3 end of the annealed primer

synthesized DNA is denatured at high temps teh temp is lowered and more primers anneal to the new strands

cycle repeated 30 times

Question 3

Techniques for the analysis of gen expression

Answer 3

• RNA isolated from sample
• RNA converted by the use of specific primers directed to a specific gene and RNA–dependent-DNA oolymerase

Question 4

PT-PCR (reverse transcription-PCR)

Answer 4

use of the produced DNA in a PCR reaction with Taq polymerase and the specific primers directed to a specific gene

Question 5

RNA sequencing quantitative technique

Answer 5

• rna is isolated from a sample and converted to DNA by the use of random primers and RNA-Dependent-DNA polymerase
  – break the produced DNA in small 200bp pieces
• sequence the dna by a massive parallel dna sequencing
  – the produced sequences are analyzed by a software and aligned to the sequence olf the genome
• teh number of sequences of that align to each locus in teh genome are quanitfied and then plotted
• resolution in the plot is very high sometimes within a base
• the plot is giving a quantitative presentation of the levels of transcrpition at each position of the genome

Question 6

antibodies are natural immunoglobulins produced by animals to combat..

Answer 6

exogenous proteins of any kind

Question 7

each b-lymphocyte produces ______ against an exogenous protein (antigen)

Answer 7

one unique antibody

Question 8

the antibody produced by one b-lymphocyte (one clone) is called

Answer 8

monoclonal antibody

Question 9

in animal blood with multiple b-lymphocytes produce multiple antibodies called…

Answer 9

polyclonal antibodies

Question 10

antibodoes consist of

Answer 10

2 heavy chain and 2 light chains joined by disulphide bonds. come together to form the antigen binding site.

Question 11

antigen binding site binds to the ____ of the ____

Answer 11

epitope
antigen

Question 12

how to produce antibodies against a desired antigen in animals

Answer 12

• inject animal with an antigen of choice and the animal will respond by producing multiple antibodies
• take blood out of these animals and prepare polyclonal antibodies against the antigen
• isolate single clones of b-lymposytes, maintain them in culture to produce monoclonal antibodies
• now we have highly specific antibodies against a protein of interest.

Question 13

techniques with antibodies

Answer 13

• immuno-flouresence
• immuno-precipitation
• ChIP chromatin immunoprecipitation

Question 14

immuno-fluorescence

Answer 14

use specific antibodies coupled to a fluorescent dye and localize the antigen in the cell

Question 14

immuno-precipitation

Answer 14

hook teh antibodies to large beads and mix the with extract, then wash away the extract. the antigen and its proteins remain with the beads via the antibody

Question 15

ChIP chromatin immunoprecipitation

Answer 15

a combination of immuno-precipitation, PCR and DNA sequencing to detect the binding of specific proteins to specific DNA sequences in vivo

Question 16

ChIp with anti-RNA pol 2 andtibodies

Answer 16

step 1: treat living cells or tissues witha membrane-permeating cross-llinker such as formadehyde
step 2: sonicate to shear cellular chromatin to short fragments and add antibody to pol 2
step 3: immunoprecipitate to isolate pol 2 cross-linked to DNA
step 4: reverse cross-linking, isolate DNA, and subject to massively parallel DNA sequencing