Techniques for the Analysis of Gene Expression Flashcards
what is the massive parallel DNA sequncing technique
sequences billions of fragments of DNA at a time
describe the steps in PCR
DNA
Ligate linkers
denature, anneal to primers
DNA synthesis
denature and wash –> single strand
anneal –> bind DNA to substrate
DNA synthesis –> make identical clusters of DNA
describe the addition of fluorescence
cut one DNA strand, denature, wash, leaving single strand
add new primer, then fluorescently labeled dNTPs, one dNTP binds, wash away excess
use fluorescent imaging to determine which bound
chemically remove fluorophore and wash
repeat until replicated
describe the cDNA library
assembling whole genome sequences
unknown genome –> create aligned library of cDNA –> sequence ordered fragments –> read sequence in order dictated by clone map –> genomic sequence
describe shotgun sequencing
assembling whole genome sequences
unknown genome of interest –> RANDOM library of cDNA –> sequence unordered fragments –> align sequenced clones by computer
what is PCR?
amplifying DNA by repetitive cycles of denaturing and renaturing of DNA in the presence of thermostable DNA polymerase
need 2 primers to anneal at 50-60 degrees
taq polymerase synthesizes new strands of DNA starting at the 3’ ends pf the annealed primers (72 degrees)
newly synthesized DNA is denatured at high temps (95)
cycled repeated 30 times
what type of technique if qRT-PCR?
QUANTITATIVE
what is RT-PCR?
REVERSE TRANSCRIPTION
by quantifying the DNA produced by PCR you indirectly quantify the abundance of corresponding RNA
how does RNA sequencing work?
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
produced sequences are analyzed by software and alined
number of sequences that align are quantified and plotted
resolution is very high
what does the RNA sequencing plot give you?
QUANTITATIVE presentation of the levels of transcription at each position of the genome
what are antibodies?
combat invading proteins
specialised class of B-lymphocytes rearrange the lg genes
each B-lymp produces ONE UNIQUE ANTIBODY
the antibody produced by one B-lymph is called MONOCLONAL ANTIBODY
POLYCLONAL ANTIBODIES = multiple B-lymphs
describe the structure of an antibody
heavy chain (middle, top)
light chain (underside)
variable region (top)
constant region (bottom)
antigen binding site (sides)
hypervariable region (top)
what does the antigen binding site recognize?
epitopes
what are epitopes?
linear, continuous sequence of AAs
conformtional made up of AAs close together in the folded protein
how do we produce antibodies?
inject an animal with a protein (antigen)
animal will respond by producing antibodies
remove blood from animal and purify immunoglobulins –> POLYCLONAL antibodies
isolate single clones of B-lymps –> MONOCLONAL
result: highly specific antibodies
what is immunofluorescence?
use specific antibodies coupled to a fluorescent dye and localize the antigen in the cell
what is immunoprecipitation?
hook the antibodies to large beads and mix with extract, was extract
antigens remain associated with beads
what is ChIP?
chromatin immunoprecipitation
combination of immuno-precipitation, PCR, and DNA seq to detect binding of specific proteins in vivo
describe the steps in immunofluorescence
prepare sample on slide
incubate with primary antibodies
wash away unbound antibodies
incubate with fluorochrome-conj secondary antibody
mount specimen and observe in fluorescent microscope
describe the steps in immuno-precipitation
primary antibody is added to mixed protein solution
agarose beads added –> form complex with Ab antibody
centrifuge and wash to separate complex from mix
elute antigen
western blot
describe the steps in ChIP
treat living cells/tissues with a membrane-permeating cross linker
sonicate to shear cellular chromatin to short fragments and add antibody to Pol II
immunoprecipitate to isolate pol II cross-linked to DNA
reverse cross-linking, isolate DNA, subject to massively parallel DNA sequencing
