Detection and quantification of NA (1)

What feature of nucleic acids is exploited to make a molecular probe?

Their capacity to base pair (Watson-Crick) with its complement

Kinds of molecular probes (2)

1. labelled ss oligonucleotide

2. PCR DNA probes

How are ss oligonucleotides labelled ?

Polynucleotide kinase transfers the gamma-phosphate of ATP to the 5’ end

How are PCR probes labelled?

Incorporation of fluorescent or alpha-phosphate dNTPs 

Important step before using a PCR probe?

Denaturation of the product so that it can be ss and bind to its complement

1st step of analysis

Cleavage by restriction enzyme

2nd step

Electrophoresis now that the segments can go through

3rd step

Denaturation of DNA through alkaline solution

4th step

Transfer to solid state support (+ve: nylon, nitrocellulose) through capillary action

Differences for RNA (2)

• 1st step= Denaturation before electrophoresis so no secondary structures

• 3rd step= denaturing buffer to stop x2

DNA/RNA bound to nitrocellulose sheets

Permanent record that be used to test probes

Amount of light/fluorescence is…

detected through autoradiography and is proportional to amount of copies in sample

Southern analysis

For DNA only

1 line in southern blot=

1 polymorphism = 2 identical alleles (homozygote)

2 lines=

Different fragment sizes = different alleles (heterozygote)

Detection without probes

If EcoRI mutated = gene size equal to non digested size

Northern analysis

For RNA only

2 lines in mRNA=

Alternative splicing or promoter

What does a northern analysis allow?

Temporal/stage and tissue specific expression

Quantitative PCR (qPCR) or RT-qPCR

Time (number of cycles) it took to reach a certain amount of fluorescence for ONE mRNA sequence

Exponential phase

Product accumulates and becomes substrate again

cDNA libraries

Evaluation of multiple mRNA

mRNA tail

3’ poly A tail 

Primer

Oligo-dT primer (complementary to tail)

ss cDNA is obtained by removing the RNA with…

Alkali or RNaseH

ss cDNA tail

Poly(dG) tail

Primer #2

Oligo-dC primer

Polymerase

E. coli DNA Pol I

Affinity column

Lined with oligo-dT primers

RNA-seq

Adaptors flank the cDNA + Computerize in genome (highly expressed = lots of RNA)