DNA Labelling - Lecture notes 10 PDF

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Notes on lecture delivered by David Groth...

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DNA Labelling DNA Probe One of the most powerful methods in molecular genetics is the detection of DNA molecules by a procedure called hybridisation As DNA molecules are not visible to the naked eye, they are labelled with a reporter group The reported group can be:  Radioactive (32P, 33P, 35S, 14C, 3H)  non-radioactive (fluorescent or chromogenic) A labelled DNA molecule is called a probe. Radioisotope Labelling

O HO P 

BASE

O

O (5') O P O P OCH  

O

OH

OH

Common Reporter Isotopes Used Label

Detail

Application

32P

Strong beta emitter

Southern hybridisation,

35S

Weak beta emitter

colony/plaque hybridisation DNA sequencing

3H

Gamma emitter

In situ hybridisation

Methods Used to Label DNA Two general approaches; 1. Incorporation of reporter molecule into the DNA either directly or indirectly, synthesis using a DNA polymerase based methods:  Primer extension  Nick Translation  Random Priming 2. End labelling methods  T4 polynucleotide kinase  Terminal transferase  End fill with DNA polymerase

Nick Translation Target dsDNA

A nick with a 3Õ-OH terminus has been introduced by Dnase I into the DNA duplex The first nucleotide on the 5Õ-P side of nick has been removed

The 5-3 exonuclease and the 5-3 polymerase plus dNTP's one of which is labelled

A nucleotide has been inserted to replace the one removed. The nick has been translated one position along the chain in a 5Õ to 3Õ directi

The nick has now translated 4 positions along the DNA chain. The process continues

Random Priming Target DNA

Denature and add random hexamer primer

Add dNTP's, labelled dNTP and Klenow DNA polymerase

Denature

End Labelling (PNK) Chemically synthesised oligonucleotide has a 5'-OH group Other DNA molecules (ds or ss) 5'-P can either be converted to a 5'-OH using Alkaline phosphatase or the exchange reaction can be used 1. Forward reaction 5'-HO

32P-ATP, Mg 2+ Polynucleotide kinase 5'-32P

2. Exchange reaction

5'-P

32P-ATP, Mg 2+, excess ADP Polynucleotide kinase 5'-32P

End Labelling (TT)

Add terminal transferase and labelled dNTP (  32PdCTP)

More nucleotides are added until the reaction is stopped

End Labelling (5’ Overhang) eg. BamH1 5’-GATCC-3’ 3’

G-5’ dATP/dTTP/dGTP or dCTP + klenow DNA polymerase) or T4 DNA polymerase

5’-GATCC-3’ 3’-CTAGG-5’

Non-Radioactive Labelling Fluorescent labelling:  Fluorescence is the “creation of an excited electronic singlet state by optical absorption and subsequent emission of fluorescence”. (Molecular Probes: the handbook. Invitrogen)  In simple terms, a photon of light striking a molecule can be absorbed and the energy from that photon increases the energy state of at least one electron within the molecule.  This “excited” state is unstable and the electron falls back to its normal energy state when the extra energy is emitted as another photon of light (usually at a longer wavelength than the original excitation photon) Fluorescent Labelling

Chromogenic Labelling Chromogenic (literally “colour forming”) tags are used to label hybridization probes. This system uses antibodies to recognise a tag that has been introduced into the probe, and to bind to those tags. The antibody is attached to an enzyme such as alkaline phosphatase that can perform a cleavage reaction on a specific compound, from which the cleavage releases an insoluble coloured product. As a result, DNA bound to a hybridization membrane shows up by the colour from the cleavage product. Apart from eliminating the need to handle radioactive compounds, chromogenic labelling has other advantages

Enzyme Chemical Luminescence (ECL)

Chromogenic Reporter

Digoxigenin reporter group

Digoxigenin dUTP

Biotin 16 dUTP

Biotin reporter group...