Lecture 3

Question 1

What is PCR?

Answer 1

Polymerase chain reaction

Question 2

What is the process of PCR?

Answer 2

Amplifying DNA by repetitive cycles of denaturing and renaturing of DNA in the presence of thermostable DNA polymerase
need two timers that anneal to the ends of the amplified dna fragment at 50-60 C, Taq polymerase synthesizes new strands of DNA starting at the 3’-ends of the annealed primers, newly synthesized dna is denatured at high temp, the temp is lowered and more primers anneal to the new strands, the cycle is repeated 30 times, if you start with one molecule of dna, you will have 2^30 molecules

Question 3

What is RNA sequencing?

Answer 3

RT-PCR (Reverse transcription-PCR) - use the produced dna in a PCR reaction with taq polymerases and the specific primers directed to a specific gene, RNA is isolated from a sample, rna is converted to DNA by the use of specific primers directed to a specific gene and rna dependent dna pol

Question 4

What are the steps to rna sequencing?

Answer 4

The produced dna is broken in small 200 bp pieces
The dna is sequenced by massive parallel dna sequencing
The produced sequences are analyzed by a software and aligned to the sequence of the genome, the number of sequences that align to each locus in the genome are quantified and then plotted, plot os a quantitative representation of the levels of transcription at each position of the genome

Question 5

What does the peak in the RT-PCR graph show you?

Answer 5

The presence of DNA pol II which usually indicates transcription

Question 6

What are antibodies?

Answer 6

Natural immunoglobulins produced by animals to combat invading exogenous proteins of any kind

Question 7

What are B-lymphocytes?

Answer 7

Rearrange the Ig genes, each b-lymphocyte produces one unique antibody against an exogenous protein (antigen)

Question 8

What does each B-lymphocyte produce

Answer 8

One unique antibody against the exogenous protein the antigen

Question 9

What is an Antigen?

Answer 9

Exogenous protein that invades the cell

Question 10

What do B-lymphocytes do when an antigen invades the cell?

Answer 10

They recognize the antigen and proliferate and produce large amount of antibody to destroy the antigen

Question 11

What is a monoclonal antibody?

Answer 11

The antibody produced by one B-lymphocyte (one clone)

Question 12

What is a polyclonal antibody?

Answer 12

Multiple B-lymphocytes that produce multiple antibodies

Question 13

What is the structure of the antibody?

Answer 13

Constant region - communicates with immune cells
Variable region - recognize a unique antigen
The antibody is built by 4 polypeptides, which have two heavy chains and one light chain

Question 14

What happens at the two regions of the antibody at the immunoglobulin gene

Answer 14

The variable regions of the immunoglobulin genes undergo chromosomal rearrangements, from the V D and J segments, they each randomly pick one to move to the variable chain, the constant chain has only a C segment, from which only one is retained to be in the constant region

Question 15

How to produce antibodies against an antigen?

Answer 15

Trick the animal by injecting it with a protein/antigen of choice. The animal will respond by producing multiple antibodies against the antigen, take the blood from the animal, purify the immunoglobulins and prepare polyclonal antibodies against the antigen, we can isolate single clones of B-lymphocytes maintain them in culture to produce monoclonal antibodies

Question 16

What are the three techniques with antibodies?

Answer 16

1. Immuno-fluorescence
2. Immuno-precipitation
3. ChIP (Chromatin Immunoprecipitation)

Question 17

What does immunoglobulin-fluorescence do?

Answer 17

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

Question 18

What is immuno-precipitation?

Answer 18

Hook the antibodies to large beads and mix them with extract, then wash away the extract. The antigen (and its associated proteins) remain associated with the beads via the antibody

Question 19

What is ChIP?

Answer 19

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

Question 20

What are the steps to immuno-fluorescence?

Answer 20

1. Prepare sample and place on microscope slide
2. Incubate with primary antibody wash away unbound antibody - primary antibody produces an antigen
3. Incubate with fluorochrome conjugated secondary antibody; wash away unbound antibody

Question 21

What are the steps to immuno-precipitation?

Answer 21

1. Primary antibody is added to mixed protein solution
2. Agarose beads are added which form a complex with a b-antigen
3. Centrifuge and wash to separate antigen complex from mix
4. Elite antigen (protein of interest) and detect via western blot

Question 22

What are the steps the ChIP?

Answer 22

1. Treat living cells/tissues with a membrane-permeating cross-linker such as formaldehyde, this penetrates the cell membrane, and interacts with amino groups which causes covalent bond from protein to DNA in vivo
2. Sonicate to shear cellular chromatin to host fragments and add antibody to pol II
3. Immunoprecipitate to isolate pol II cross-linked to DNA
4. Reverse cross-linking isolate DNA, and subject to massively parallel DNA sequencing, release dna from immuno-precipitate

Question 23

What do the four graphs from the ChIP seq with different antibodies show you?

Answer 23

CTCF: regions bound by the insulator protein CTCF
RNA POL II: regions bound by RNA pol II
H3K27me3: regions bound by histones, the peaks is where the dna is wrapped in histones, meaning no tln