Oligonucleotides – short single-stranded DNA fragments
– have defined sequences
– can be synthesized chemically
uses – probes for hybridization
– PCR/sequencing primers
– site-directed mutagenesis
notes on synthesis – multiple repeated steps
– easy to automate
– custom oligos usually ordered from supplier
– ~ $50 per standard (20-30 nucleotide) oligo
– may be up to 150 nucleotides – but most are < 50
– longer sequences hard to synthesize
– large number cycles
– greater chance of mistakes
– e.g. n + 1, n – 1 products
– can incorporate modified bases – e.g. inosine
– allow different hybridization properties
– synthesis is 3' to 5'
– 3' PO4 of nucleotide reacts with 5' OH on chain end
– opposite from enzymatic synthesis
– need blocking groups to prevent side-reactions
– on amino groups of nitrogenous bases
– on 5' OH
– on – 3' PO4
– hydrolysed off during or after synthesis
protocol
0. attach 3' OH of first nucleotide to glass beads in column
– allows excess reagents to be washed out of column
1. hydrolyse blocking group from 5' OH
– wash out of column
2. add next nucleotide
– activated 3' PO4 reacts with 5' OH of first nucleotide
– forms phosphite linkage
– wash excess nucleotides out of column
3. “cap” unreacted 5' OH with acetylating reagent
– prevents (n – 1) products
– i.e. not reacted in 1 cycle, reacted in next
4. oxidize phosphite linkage to phosphate
– product ready to enter next cycle
alternate protocol – uses light-sensitive 5' blocking group
– removed by laser
– use programmed “masks”
– control activation of 5' ends
– can synthesize different oligos at once
– in same reaction mix
– e.g. different regions of oligonucleotide array chips
synthesis of double-stranded DNA
– from hybridization of complementary oligonucleotides
– e.g. 2 strands of multiple cloning sites
– mixed & cloned in vectors
– longer sequences (e.g. synthetic genes)
– use oligos with complementary 3' ends
– hybridize with short double-stranded region
– extend 3' ends with DNA polymerase
(5' to 3')
– join ends with ligase