RR1 Flashcards
name methods of qualitative analysis
nature of molecules in question
size
nt composition
conformation/configuration
Structure
types of molecules - isoforms and variants, knowing proteins- help develop drugs
name methods of quantitative analysis
levels of gene products
tumour markers
describe molecular probes
nucleic acid that can waston crick base pair with sequence of interest
describe process of molecular probes
complex mixture of macromolecules –> binding to membrane (nitrocellulose/nylon), separate via agarose gel, transfer to solid state - permanent record –> probe specific for target (find needle in haystack, hybridize blot with probe) –> remove nonspecific by washing, favours specific interaction –> target detection, visualize it
single stranded oligonts can be labelled using
Polynucleotide kinase = pnk
make oligont with reverse complementary sequence so will base pair
5’ end radioactive isotope = labelled ogliont
pnk isolated from t4 phage
explain PNK
phosphorylate nucleotides by transferring the gamma phosphate of ATP to free hydroxyl at 5’ end of the synthetic oligonucleotide (Since it does not have ones)
isotope phase = Radioactive phosphate will transfer and make oligonucleotide Radio active
Run gel filtration column to get rid of the atp then you have purified probe
what can pcr do
used to make labeled dna probes
for dsdna
label must be rendered single stranded and then transferred to membrane for analysis
name solid state supports
nylon or nitrocellulose
describe southern blot
DNA - must be denatured, put in agarose gel in alkaline solution
make sure it stays denatured
transfer to stable support and forms permanent record
describe northern blot
denature rna first and maintain in gel so will not refold and transfer to membrane
describe what happens after solid state support for blots
covalently bound and levels and positions are permanently recorded
can be hybridized with probe to any sequence of interest
washes remove non-specific
only complementary sequences are left on blot following autoradiography
what is difference between northern, southern and western blots
dna = southern
rna = northern
protein = western
how can you tell level - for blots with probes
intensity of probe binding
x ray for radioactive probe and tells you where nucleic acid target is in that separation
describe detecting polymorphisms with probes
quantitative info
cut with restriction enzymes - ecor1 cuts 2 fragments - one big and small
if mutation - variation disrupts ecor1 - visible since probe recognizes one giant fragment only
what is RFLPs
restriction fragment length polymorphisms
what used to be used for pedigrees
RFLPs
using dna-southern blot
describe detecting polymorphisms without probes
simple pcr if know sequence
brca 1 and 2 = look for variation around sequence by amplifying
should see different fragment sizes in agarose gel
if not = mutant allele, ecor 1 did not cut
describe Nucleic acid hybridization techniques enable RNA detection - Northern analysis
nucleic acids of complementary sequences can base pair to form double stranded hybrid = denatured,
Ran in gel & transferred then
detected we single stranded DNA probe
do not need to cut rna before
levels change - says something of level of gene expression or stability of rna
what info can northern analysis give
tissue expression
stage specific temporal expression
quantitative = asses abundance of rna from rna blotting, where rnas are expressed
qualitative = understand various isoforms that are largely dependent on alternative splicing reactions that take place within tissues or on a temporal scale
do the probes have to be super specific for northern blot
no
not super specific to mrna
do not have to wash hard
describe RT-PCR
rt converts mrna to cdna –> pcr reaction that includes intercalating fluorescent due - emits signal when incorporated into growing dna polymer (more dna produced = more fluorescence)
quantifies level of specific transcript
what primers can be used for rt-pcr
poly dt primer = will prime all mrnas - since poly a tail then can extend ssdna from primer
one specific mrna primed - prime synthesis of dna in an rt reaction
what do all pcr reactions go through
exponential phase
linear phase
reaches plateau
more cnda in original sample = faster plateau is reached
describe general pcr curve
product becomes substrate
getting to threshold = depends how much substrate start with
plateau = reaction done
what is critical point on pcr curve
where becomes exponential - quantification cycle
amplification goes above threshold
can estimate original amount of dna
good representation of single thing but not a good global view of isoforms
describe making cdna libraries
mrna converted to cdna by priming poly a tail with single stranded poly t oligont –> rt uses primer and initiates single strand synthesis –> rna then removed and add poly dg adapter to 3’ end –> poly dc primer used to initiate synthesis of second dna strand (rna-dna dihybrid, treat with rna’s h enzyme or alkali to separate) –> dna pol 1 extends second strand –> makes double stranded –> all cdnas have same ends, then can clone those into bacterial vectors and use whenever
what is a cdna library
dna based representation of all different mrnas present in given sample
permanent collection of sequences of all mrnas that were present in given sample and abundance - generally accurate
describe poly dg and poly dc primers for cdna library
Add poly dg primer
using lots of ligase- adapt it and put Oligonucleotide
with only a series DGs=
Generate 3’ end
same for all cDNAs
then use polyDC primer and prime 3’ end Synthesis of a second strand of DNA complementary
describe RNA-seq
new method - new role of cdnas
rna -seq = next generation sequencing method coupled with formation of cdna libraries
purification of rna from some tissue (for mrna = do affinity chromatography, with poly t column = enrich for poly a containing mrnas) –> using rt make cdna library –> ligation of adaptors for ngs, carry out sequence analysis –> genome alignment and quantification, take sequences and align to genome map or with sequences and get reads
number of times you get a specific sequence read = corresponds to abundance of cdna in that reaction
what does rna-seq provide
wide view of all genes in genome
global gene expression
global view
not as accurate as rtpcr or northern blots
what is capillary action
pull water soluble molecules up from gel –> blot
what is amplicon
big amount of dna from amplification
