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