Transcript Making probes/primers

Labelling probes and primers
•
In the cases of Northern and Southern blots probes are pieces of
single stranded DNA that are complimentary to the single stranded
target on the membrane.
•
Short oligonucleotides or primers are used for labeling sections for
light, fluorescent or electron microscopy and also in amplification
reactions
How are probes made?
1.
synthetically
2.
cDNA derived from RNA by using reverse transcriptase
3.
Isolated from genomic DNA using PCR or restriction digest
followed by PCR
4.
Isolated from many copies of plasmids following digestion and gel
isolation
5.
Isolated from a specialized viral vector that makes single stranded
DNA
•
•
Probes and primers vary in size from 20 to 1000s of bases
Probes for Southerns tend to be at least 100bp long
DNA synthesis using the Phosphoramidite
method.
•Before the start of synthesis amino groups of adenine, guanine
and cytosine are derivatised by addition of benzoyl, isobutyryl
and benzoyl groups respectively to prevent undesirable side
reactions during chain growth.
•Thymine is not treated as it has no amino group
•Solid phase synthesis
•Initial nucleoside (base plus sugar only) is attached to an inert
support by its 3’ terminus via a spacer molecule.
•The spacer molecule is attached to the inert support by
covalent bonding
•This nucleoside will ultimately be the 3’terminal nucleotide of
the synthesised strand
Only useful for pieces
up to about 100 bases
• The solid support is loaded into the reaction column.
• In each step, the solutions will be pumped through the
column.
• The reaction column is attached to the reagent delivery
lines and the nucleic acid synthesizer.
• Each base is added via computer control of the reagent
delivery
• Initial nucleoside (base
plus sugar only) is
attached to an inert
support by its 3’
terminus via a spacer
molecule.
• The spacer molecule is
attached to the inert
support by covalent
bonding
• After the initial
nucleoside is attached
to the column the cycle
begins (see flow chart)
Phosphoramidite molecule
• A dimethoxytrityl group DMT has been attached
to the 5’terminusnof the initial nucleoside to
prevent the 5’ hydroxy from reacting non
specifically
• Each nucleoside to be added has a 5’ DMT and
a diisopropyl amine group attached to the 3’
phosphite group which is itself protected by a
methyl residue.-phosphoramidite
Steps in the cycle
1. Washing
• Acetonitrile or some other anhydrous reagent
is used to remove any water that may be
present in the column
• column flushed with argon to purge the
acetonitrile
2. Detitrylation
• 5’ DMT group removed from the attached
nucleoside by treatment with trichloroacetic
acid (TCA) to give a reactive 5’ OH group
3.Washing
• Acetonitrile or some other anhydrous reagent is
used to remove TCA
• column flushed with argon to purge the
acetonitrile
4.Activation and Coupling
• The next prescribed base introduced as a
phosphoramidite and tetrazole is introduced at
the same time
• Tetrazole activates the phosphoramidite so that
its 3’ phosphite forms a covalent bond with the 5’
hydroxy of the initial nucleotide
5. Washing
• Unincorporated phosphoramidite and tetrazole are
removed by flushing with argon
6. Capping
• not all the attached nuleosides are linked to a
phosphoramidite during the first coupling so unlinked
residues need to be capped to prevent them from linking
to the next incoming nucleotide in the next cycle
• acetic anhydride and dimethyaminopyridine are added to
acetylate the unreacted 5’ hydroxyl groups
• 7. Oxidation
• the internucleotide link is a phosphite
triester bond which is unstable and prone
to breakage by acids and bases
• it is oxidised with an iodine mixture to form
a stable pentavalent phosphate triester
8. Back to the start of the cycle to add the
next nucleotide
9. Cycling end
• When the oligo is complete i.e. the final cycle is
complete each phosphodiester still has a methyl group
and every G, C and A still carries an amino protecting
group and the 5’ terminus of the final nucleotide has a
DMT group
• Methyl groups are removed by chemical cleavage while
still on column
• DNA oligonucleotides are cleaved from column at the
3’hydroxyl terminus and DNA is eluted from column
• Amino protecting groups of bases are stripped
• DNA is detritylated
• The DNA oligonucleotides are phosphorylated at the 3’
end by a kinase and ATP
OLIGONUCLEOTIDES ARE NOW READY TO ROCK!!!!!!