Lecture 3 Flashcards
What is gene expression?
The information in a gene is used to create a functional protein
What is Polymerase Chain Reaction (PCR)?
Creating clusters of identical DNA attached to a solid support
What is the purpose of PCR?
To amplify DNA
What does denaturing do to a protein?
It makes it single stranded
How does PCR work?
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
What are ligate linkers?
Short DNA molecules attached to the end of DNA
What technique can you use to figure out sequence a DNA strand is?
Fluourescently labeling dNTPS
How does fluroescently labeled dNTPs work?
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
What are the two strategies for assembling whole genome sequences?
1.) cDNA library
2.) Shotgun sequencing
What is a cDNA library?
Create a aligned library of cDNA, sequence ordered fragments, read sequence in order dictated by clone map
What is shotgun sequencing
1.) Create a random library of cDNA
2.) Sequence unordered fragments
3.) Align sequenced clones by computer
What is PCR using?
Is the presence of amplifying DNA by renaturing and denaturing DNA using DN Polymerase (Taq polymerase)
What do you need to anneal to the ends of the amplified DNA?
Two primers between 50-60 degrees
What does Taq polymerase do?
Synthesizes new DNA at the 3’ end of the annealed primers at 72 degrees
What happens to the new synthesized DNA?
It gets denatured at high temperatures (95 degrees)
What happens when the temperature gets lower (50-60 degrees)
New primers will anneal to the new strands
How many times is PCR repeated?
30 times - this means ion you start with two molecules, after PCR you will have 2^30 molecules.
What type of technique is RNA sequencing and qRT-PCR?
Quantitative techniques used for gene expression
What happens during RNA sequencing for the analysis of gene expression?
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
What is RT-PCR?
Using the produced DNA in a PCR reaction with Taq polymerase and the specific primers directed to a specific gene
What does quantifying the DNA produced by PCR do?
You are indirectly quantifying the abundance of the corresponding RNA in the sample.
What happens during RNA sequencing for the GLOBAL analysis of gene expression?
RNA is isolated from same and converted into DNA using random primers and a RNA dependent DNA polymerase
What are you going to do with the produced DNA?
Break it into small 200 bp pieces
How are you going to sequence the produced DNA?
By Massive Parallel DNA sequencing
What happens to the produced sequences?
They will be analyzed by software and aligned to the sequence of the genome.
What happens to the number of sequences that align at each locus in the genome?
Are quantified and potted
What is the plot demonstrating?
A quantitative presentation of the levels of transcription at each position on the genome.
What are antibodies?
Natural immunoglobins produced by animals to combat invading exogenous proteins of any kind.
What do a special case of lymphocytes do?
Rearrange the Ig genes
What does each B-lymphocyte produce?
One unique antibody against an exogenous protein (antigen)
What happens upon invasion of an antigen?
The B-lympohocytes recognize this antigen, proliferate and produce large amounts of the antibody to destroy it.
What is the antibody procured by one B-lymphocyte called?
Monoclonal Antibody (only attacks one thing)
What are multiple B-lymphocytes that produce antibodies called (blood)?
Polyclonal Antibodies
What do antibodies contain?
A heavy region and a light chains
How are the heavy and light chains joined together?
By disulphide bonds
What do each heavy chain and light chain consist of?
Constant Domain and the Hypervariable Domain
What does the hyper variable domain of the heavy and light chain contain?
The antigen binding site
What do the variable region of the Ig gene undergo?
Chromosomal rearrangement
What can the heavy chain be encoded up to?
100-V, 30-D and 6-J
What is the Antigen binding site called?
Paratope
What binds to the Antigen Binding Site
Epitope of the Antigen
What can epitopes be?
Linear, made up of chains of amino acids
Conformational, made up of amino acids close together in the folded structure of a protein
How can you test antibodies on an animal?
Can inject it with a protein (antigen) and the animal will respond by producing multiple antibodies against the antigen
What can you do with the animal after the antibody has been produced?
Take out the antibodies from the blood, purify the immunoglobulins and prepare polyclonal antibodies against the antigen
How can you produce monoclonal antibodies?
Isolating single clones of the B-lymphocytes and maintaining them in culture
What are B cells?
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.
How does immunization of a rabbit work?
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
What is Immuno-Fluroscence?
Take an antibody, couple it with a fluorescent dye and localize the antigen within a cell.
What is Immuno-Precipitation?
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
What is Chromatin-Immuno Precipitation?
It is a combination of immuno precipitation, PCR, and DNA sequencing to detect the bonding of specific proteins to specific DNA sequences in vivo
What are the step of Immuno-Fluorence?
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.
What are the steps of immuno-precipitation?
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.
What is Step 1 of ChIP
Treat living cells or tissues with a membrane-permeating cross linker such as formaldehyde
What is Step 2 of ChIP?
Sonicate to shear cellular chromatin into short fragments and add antibody to RNA pol II
What is Step 3 of ChIP?
Immunoprecipate to isolate Pol II that is cross-linked to DNA
