what is a DNA oligonucleotide?
what are important structural features?
string of 18-25 ntds that's complementary to a sequence on the mRNA
serves as a "probe" for an mRNA containing the target sequence
5' end: radioactive phosphate group or another detectable chemical group
what is the purpose of different experimental methods looking at nucleic acid synthesis?
diff experimental methods to probe for transcriptomes or genomes
what is the purpose of Northern Blotting?
detect a specific part of an RNA out of all the mRNAs
what is the transcriptome?
the whole set of RNAs a cell expresses in a particular situation
different cells have different transcriptomes
what kind of RNA is used in northern blotting? why?
cytoplasmic mRNA because has poly-A tail which makes it localizable
nuclei are removed to avoid contamination w/ pre-mRNA
in northern blotting, how is isolated poly-A+ mRNA treated?
pass it through an oligo-dT column
get A-T complementary base pairing
poly-A tail sticks to oligo-dTs, rest of mRNA is washed away
in northern blotting, what happens to the poly-A+mRNA w/ its oligo-dT tail?
load into a gel, separates by size
then blot the gel onto a membrane and apply a probe that binds to target mRNA sequences by complementarity
wash filter; expose to X-ray film; see band pattern
what is the probe in Northern blot?
a single-stranded DNA that's radioactively labeled at its 5' end
sequence is complementary to sequence known to be on target mRNA
what information do we get from Northern blotting?
1) tissue-specific gene expression
2) regulated expression
3) alternative splicing
4) intsertion/deletion mutations
5) defective splicing
6) quantitative info about level of expression, b/c amount of probe that binds is function of number of target molecules present
how can northern blot tell us about tissue specificity?
glucose in liver vs glucose in brain is good example-
the more mRNA in gel, the more probe binds, the more intense the signal is
so see from gel tissue specificity where diff genes are expressed differentially based on their location/function
what is the purpose of doing PCR?
amplify a specific DNA fragment from a complex mixture
what is the order of the first steps of PCR, before amplification?
1) heat mixture to separate DNA strands
2) add 2 specific oligonucleotide primers- complementary to short stretches on eithe rside of desired fragment
3) lower temperature
primers hybridize to DNA at desired ends of target sequence
4) add heat stabled DNA polymerase & nucleotide triphosphates
what does heat-stablized DNA polymerase do in PCR?
extends the primers, synthesizes new complementary DNA strands
what is produced at end of 1st cycle of PCR?
2 double stranded DNA molecuels containing target sequence
how many double stranded DNA molecules at end of 2nd PCR cycle? 3rd?
describe the process of controlling the PCR tube reaction conditions and what happens at each step?
1) denature at 95 degrees C
2) extension by adding heat-stablized DNA polymerase which extends from 3' end of primer at 72 degrees C
3) anneal to allow specific primer/target hybridization/binding at 50-60 degrees C
what is the basis of PCR?
primer specificity - using an oligo primer w/ a 20-ntd sequence that's complementary to a known 20-ntd sequence will only bind to particular sequence of interest
how can we be confident PCR works properly?
use 2 oligos complimentary to sequences known to be located a certain distance from each other
there's virtually 0% chance that you find anything except for the exact localized pair of ~20-ntd sequences 300 base-pairs apart from each other that you wanted to find
how does primer extension work in PCR?
3' OH is free on oligos
have dNTPs and a polymerase in test tube
polymerase in response to DNA template and 3' OH of oligos will extend primers in each direction, amplifying # of copies w/ each round of PCR
what are the diagnostic uses of PCR?
1) detection of carrier for genetic diseases
2) single nucleotide polymorphisms, SNPs
3) PCR for RNA target
how does PCR for point mutations/indels work?
use PCR to amplify a particular chromosomal DNA segment w/ known DNA mutations
what is a restriction endonuclease?
enzyme that cuts DNA in middle fo the sequence at a specific target sequence
what id Dde I?
a restriction endonuclease
when using PCR for detection of a carrier of a genetic disease,
how many bands show up on a gel if the carrier is a heterozygote?
1 for the wildtype, b/c does not have restriction endonuclease's recognition site
2 bands for where the restriction endonuclease cut the mutated sequence of interest
how is PCR used for "foreign" genome detection?
can do PCR for a "foreign" genome, such as TB's genome, rather than ours, from a sputing sample
can get immediate detection of that foreign organism's genome's presence in culture
what is real-time PCR?
follow, in real time, course of amplification of target sequence
as number of cycles of PCR reaction continues, get more and more of target DNA present which is detectable by level of flourescence (i.e. of SYBR Green) that flouresces when it has bound to target DNA sequence of interest
rate at which flourescent intensity increases is directly related to quantity of input taget sequence
how soon (time) sample rises above detection level = signaling mechanism
How can PCR be used to look at RNA? why does this need to happen?
usually, PCR works by DNA polymerase, SO
convert RNA to CDNA, a complementary copy of mRNA sequence via use of reverse transcriptase
describe process of using PCR to look at RNA genome expression
describe conersion of RNA --> cDNA
to convert RNA to cDNA:
1) put mRNAs into tube w/ oligo-dT primer, which binds to poly-A tail of mRNAs
2) put oligo-dT bound poly-A+ mRNAs into tube w/ reverse transcriptase & dNTP
3) reverse transcriptase will convert mRNA to cDNA
4) get hybrid between mRNA and cDNA
treat w/ NaOH to destroy RNA
add DNA polymerase, dNTPs
5) now do PCR w/ gene-specific primers to new double-stranded cDNA
what is this?
quick detection of a DNA sequence in a tumor cell is possible using real time PCR
see time-related increased flourescence of sequence of interest in tumor versus normal tissue indicates presence of tumor sequences in sample
how can SNPs be looked at via PCR?
1) purify out RNA
2) bind poly-A+ fraction of mRNA
bind to oligo-dT column
3) take fragments, convert to cDNA
4) do PCR of target sequence including a SNAP
5) can analyze for single nucleotide polymorphisms in a patients' genotype w/ respect to the population for a particular polymorphism
what does analyzing a person's SNPs of the ApeE gene tell us?
different polymorphic forms of the ApoE gene have been associated with risk of early onset Alzheimers;
an individual w/ E4 version, if heterozygote, has 4x risk of early onset Alzheimer's; homozygote = 10x risk
can see this on a PCR
what is Tay Sachs caused by?
30%: G-C bp switch in intron 12
70%: 4 bp insert in Exon 11