RR1

Question 1

name methods of qualitative analysis

Answer 1

nature of molecules in question
size
nt composition
conformation/configuration
Structure
types of molecules - isoforms and variants, knowing proteins- help develop drugs

Question 2

name methods of quantitative analysis

Answer 2

levels of gene products
tumour markers

Question 3

describe molecular probes

Answer 3

nucleic acid that can waston crick base pair with sequence of interest

Question 4

describe process of molecular probes

Answer 4

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

Question 5

single stranded oligonts can be labelled using

Answer 5

Polynucleotide kinase = pnk
make oligont with reverse complementary sequence so will base pair
5’ end radioactive isotope = labelled ogliont
pnk isolated from t4 phage

Question 6

explain PNK

Answer 6

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

Question 7

what can pcr do

Answer 7

used to make labeled dna probes
for dsdna
label must be rendered single stranded and then transferred to membrane for analysis

Question 8

name solid state supports

Answer 8

nylon or nitrocellulose

Question 9

describe southern blot

Answer 9

DNA - must be denatured, put in agarose gel in alkaline solution
make sure it stays denatured
transfer to stable support and forms permanent record

Question 10

describe northern blot

Answer 10

denature rna first and maintain in gel so will not refold and transfer to membrane

Question 11

describe what happens after solid state support for blots

Answer 11

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

Question 12

what is difference between northern, southern and western blots

Answer 12

dna = southern
rna = northern
protein = western

Question 13

how can you tell level - for blots with probes

Answer 13

intensity of probe binding
x ray for radioactive probe and tells you where nucleic acid target is in that separation

Question 14

describe detecting polymorphisms with probes

Answer 14

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

Question 15

what is RFLPs

Answer 15

restriction fragment length polymorphisms

Question 16

what used to be used for pedigrees

Answer 16

RFLPs
using dna-southern blot

Question 17

describe detecting polymorphisms without probes

Answer 17

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

Question 18

describe Nucleic acid hybridization techniques enable RNA detection - Northern analysis

Answer 18

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

Question 19

what info can northern analysis give

Answer 19

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

Question 20

do the probes have to be super specific for northern blot

Answer 20

no
not super specific to mrna
do not have to wash hard

Question 21

describe RT-PCR

Answer 21

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

Question 22

what primers can be used for rt-pcr

Answer 22

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

Question 23

what do all pcr reactions go through

Answer 23

exponential phase
linear phase
reaches plateau
more cnda in original sample = faster plateau is reached

Question 24

describe general pcr curve

Answer 24

product becomes substrate
getting to threshold = depends how much substrate start with
plateau = reaction done

Question 25

what is critical point on pcr curve

Answer 25

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

Question 26

describe making cdna libraries

Answer 26

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

Question 27

what is a cdna library

Answer 27

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

Question 28

describe poly dg and poly dc primers for cdna library

Answer 28

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

Question 29

describe RNA-seq

Answer 29

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

Question 30

what does rna-seq provide

Answer 30

wide view of all genes in genome
global gene expression
global view
not as accurate as rtpcr or northern blots

Question 31

what is capillary action

Answer 31

pull water soluble molecules up from gel –> blot

Question 32

what is amplicon

Answer 32

big amount of dna from amplification