Lecture 3

Question 1

What is gene expression?

Answer 1

The information in a gene is used to create a functional protein

Question 2

What is Polymerase Chain Reaction (PCR)?

Answer 2

Creating clusters of identical DNA attached to a solid support

Question 3

What is the purpose of PCR?

Answer 3

To amplify DNA

Question 4

What does denaturing do to a protein?

Answer 4

It makes it single stranded

Question 5

How does PCR work?

Answer 5

Take double stranded DNA, and attach ligate linkers to it, denature the protein, then anneal it to the primers, it will undergo DNA synthesis, then you denature again, wait for it to anneal again and then undergo DNA synthesis again

Repeat this 10x

Question 6

What are ligate linkers?

Answer 6

Short DNA molecules attached to the end of DNA

Question 7

What technique can you use to figure out sequence a DNA strand is?

Answer 7

Fluourescently labeling dNTPS

Question 8

How does fluroescently labeled dNTPs work?

Answer 8

1.) Cut one DNA strand (coming from PCR) and denature it to make it single stranded

2.) Add a new primer to it. Fluroscently label dNTP, one dNTP binds, wash away excess.

3.) Fluroscent imaging to determine which dNTP bound

4.) Chemically remove fluorphore and wash

5.) Repeat

Question 9

What are the two strategies for assembling whole genome sequences?

Answer 9

1.) cDNA library

2.) Shotgun sequencing

Question 10

What is a cDNA library?

Answer 10

Create a aligned library of cDNA, sequence ordered fragments, read sequence in order dictated by clone map

Question 11

What is shotgun sequencing

Answer 11

1.) Create a random library of cDNA

2.) Sequence unordered fragments

3.) Align sequenced clones by computer

Question 12

What is PCR using?

Answer 12

Is the presence of amplifying DNA by renaturing and denaturing DNA using DN Polymerase (Taq polymerase)

Question 13

What do you need to anneal to the ends of the amplified DNA?

Answer 13

Two primers between 50-60 degrees

Question 14

What does Taq polymerase do?

Answer 14

Synthesizes new DNA at the 3’ end of the annealed primers at 72 degrees

Question 15

What happens to the new synthesized DNA?

Answer 15

It gets denatured at high temperatures (95 degrees)

Question 16

What happens when the temperature gets lower (50-60 degrees)

Answer 16

New primers will anneal to the new strands

Question 17

How many times is PCR repeated?

Answer 17

30 times - this means ion you start with two molecules, after PCR you will have 2^30 molecules.

Question 18

What type of technique is RNA sequencing and qRT-PCR?

Answer 18

Quantitative techniques used for gene expression

Question 19

What happens during RNA sequencing for the analysis of gene expression?

Answer 19

RNA is isolated from a sample, and is converted to DNA by the use of SPECIFIC primers directed to a specific gene and RNA-dependent DNA polymerase

Question 20

What is RT-PCR?

Answer 20

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

Question 21

What does quantifying the DNA produced by PCR do?

Answer 21

You are indirectly quantifying the abundance of the corresponding RNA in the sample.

Question 22

What happens during RNA sequencing for the GLOBAL analysis of gene expression?

Answer 22

RNA is isolated from same and converted into DNA using random primers and a RNA dependent DNA polymerase

Question 23

What are you going to do with the produced DNA?

Answer 23

Break it into small 200 bp pieces

Question 24

How are you going to sequence the produced DNA?

Answer 24

By Massive Parallel DNA sequencing

Question 25

What happens to the produced sequences?

Answer 25

They will be analyzed by software and aligned to the sequence of the genome.

Question 26

What happens to the number of sequences that align at each locus in the genome?

Answer 26

Are quantified and potted

Question 27

What is the plot demonstrating?

Answer 27

A quantitative presentation of the levels of transcription at each position on the genome.

Question 28

What are antibodies?

Answer 28

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

Question 29

What do a special case of lymphocytes do?

Answer 29

Rearrange the Ig genes

Question 30

What does each B-lymphocyte produce?

Answer 30

One unique antibody against an exogenous protein (antigen)

Question 31

What happens upon invasion of an antigen?

Answer 31

The B-lympohocytes recognize this antigen, proliferate and produce large amounts of the antibody to destroy it.

Question 32

What is the antibody procured by one B-lymphocyte called?

Answer 32

Monoclonal Antibody (only attacks one thing)

Question 33

What are multiple B-lymphocytes that produce antibodies called (blood)?

Answer 33

Polyclonal Antibodies

Question 34

What do antibodies contain?

Answer 34

A heavy region and a light chains

Question 35

How are the heavy and light chains joined together?

Answer 35

By disulphide bonds

Question 36

What do each heavy chain and light chain consist of?

Answer 36

Constant Domain and the Hypervariable Domain

Question 37

What does the hyper variable domain of the heavy and light chain contain?

Answer 37

The antigen binding site

Question 38

What do the variable region of the Ig gene undergo?

Answer 38

Chromosomal rearrangement

Question 39

What can the heavy chain be encoded up to?

Answer 39

100-V, 30-D and 6-J

Question 40

What is the Antigen binding site called?

Answer 40

Paratope

Question 41

What binds to the Antigen Binding Site

Answer 41

Epitope of the Antigen

Question 42

What can epitopes be?

Answer 42

Linear, made up of chains of amino acids Conformational, made up of amino acids close together in the folded structure of a protein

Question 43

How can you test antibodies on an animal?

Answer 43

Can inject it with a protein (antigen) and the animal will respond by producing multiple antibodies against the antigen

Question 44

What can you do with the animal after the antibody has been produced?

Answer 44

Take out the antibodies from the blood, purify the immunoglobulins and prepare polyclonal antibodies against the antigen

Question 45

How can you produce monoclonal antibodies?

Answer 45

Isolating single clones of the B-lymphocytes and maintaining them in culture

Question 46

What are B cells?

Answer 46

B cells are white blood cells that produce antibodies to fight infection. They are a type of lymphocyte, which are often found in lymph nodes and the spleen.

Question 47

How does immunization of a rabbit work?

Answer 47

1.) Inject the rabbit with an antigen 2.) B cells are going to specifically respond to the antigen and produce antibodies 3.) The antibodies will enter the blood stream 4.) Isolate the B-lymphocytes to create monoclonal antibodies

Question 48

What is Immuno-Fluroscence?

Answer 48

Take an antibody, couple it with a fluorescent dye and localize the antigen within a cell.

Question 49

What is Immuno-Precipitation?

Answer 49

Hooking an antibody to large beads, mixing them with extract, washing aways the extract and having the antigen bound to the beads via the antibody

Question 50

What is Chromatin-Immuno Precipitation?

Answer 50

It is a combination of immuno precipitation, PCR, and DNA sequencing to detect the bonding of specific proteins to specific DNA sequences in vivo

Question 51

What are the step of Immuno-Fluorence?

Answer 51

1) Take a sample and put it on a microscope slide 2.) Incubate with primary antibody, wash away unbound antibody 3.) Incubate with fluorescent dye and wash away unbound antibody 4.) Observe specimen in fluorescent microscope.

Question 52

What are the steps of immuno-precipitation?

Answer 52

1.) Antibody is mixed into solution 2.) Adding the beads into the solution to form a complex with the antigen 3.) Extract and centrifuge the antigen, then wash the extract 4.) Elute the antigen and detect via a Western blot.

Question 53

What is Step 1 of ChIP

Answer 53

Treat living cells or tissues with a membrane-permeating cross linker such as formaldehyde

Question 54

What is Step 2 of ChIP?

Answer 54

Sonicate to shear cellular chromatin into short fragments and add antibody to RNA pol II

Question 55

What is Step 3 of ChIP?

Answer 55

Immunoprecipate to isolate Pol II that is cross-linked to DNA