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. synthetically2. cDNA derived from RNA by using reverse transcriptase3. Isolated from genomic DNA using PCR or restriction digest

followed by PCR4. Isolated from many copies of plasmids following digestion and gel

isolation5. 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 argon6. 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!!!!!!