nucleic acid detection and eukaryotic transcription

lec 16 - 17

what does qualitative analysis of nucleic acids tell us? (4)

• structure

• conformation

• nature/size of molecules

• nucleotide composition

what does quantitative analysis of nucleic acids tell us? (2)

• levels of gen products

• over/underexpression

general scheme of how probes work

2 simple ways of making probes

• ss oligonucleotides

• pcr

how can ss oligo-nt be used as probes?

• make ss oligonucleotide reverse complimentary sequence to target

• polynucleotide kinase phosphorylates the free OH on the 5’ end with a radioactive gamma phosphate

PNK

polynucleotide kinase

how can pcr be used to make probes?

• amplify dna that is compliment to target dna

• introduce labelled dNTPs into PCR amplified dna

  • can use radiolabelled dCTPs 

  • can use fluorescentlylabelled dNTPs

what are solid supports usually made of?

nylon or nitrocelluslose

do agarose gels need to be marked after the gel has been run?

• yes

• to use as a reference for interpreting the positions, lane numbers, and well locations of blot later

how does dna get onto a solid state support?

• run an agarose gel

  • with rna there must be formaldhyde in gel

• dna gets pulled onto membrane through capillary action

UV crosslinking

• ensures nucleic acids are permanently bound to solid state support

• once bound, level and positions are recorded

hybridization

• letting a probe interact w/ nucleic acid on a solid support then removing the membrane and washing 

• only target dna remains

autoradiography

• used to detect the target dna left behind after hybridization

• amount of radioactivity represents the number of copies in the sample

southern analysis

• dna only blotting technique

  • follows agarose gel, membrane transfer method

• probe doesnt need to bind 100%

• used for polymorphisms

• digest dna, run in gel, then denature

northern analysis

• rna only blotting technique

  • follows agarose gel, membrane transfer method

• rna doesnt need to be digested before gel

RT- qPCR

• reverse trancriptase quantitative pcr

• determines mRNA levels

• make cDNA then do PCR using a fluorescent dye

• knowing how many PCR cycles occur before Ct tells how much starting material you had 

Ct

• threshold cycle seen in PCR

• point where growth becomes exponential

• faster ct is reached, more starting mrna u have

cDNA stands for

complimentary dna

how is cdna made

• poly A tail of mrna is primed with a ss poly T oligonucleotide

• RT uses that primer to make compolimentary ssdna

• mrna is removed

• poly dG adapter anneals to 3’ ends of cDNA

• poly dC primer intiates synthesis of second strand

• e coil

rna sequencing (6 stps)

• ngs methos for studying gene eexpression

1. isolate mrna, rrna, ncrna from sample

2. used poly A selection or size selection to isolate specific rna species

3. rna —> cdna

4. ligate short dna adaptors to each end of cdna to act as primer

5. optional : pcr to amplify library

6. use ngs to read fragments and map to genome

main method of regulating gene expression

transcriptional control

3 stages of transcription

1. intiation - rna pol 2 binds to promoter, locally denatures dna, and catalyzes first phosphodiester linkage

2. elgongation - rna pol makes rna in 5’ → 3’ direction

3. temrination - rna pol sees stop siite, releases dna, and dissociates

polycistronic vs monocistronic

• polycistronic have multiple genes regulated by the same promoter

  • seen in prokaryotes

• mono had one promoter per gene

  • eukaryotic

2 key structures of rna pol 2

• clamp

• CTD with heptapeptide sequence

what is a phosphorylated ctd of an rna pol 2 associated with?

active transcription