Originally Posted by af malik
Also tell me reverse complement primer?
A few cut/paste pointers, but important nonetheless...
Often we need to obtain the complementary strand of a DNA sequence [to boost yield, I presume - NAPR
]. As DNA is antiparallel, we really need the reverse complement sequence to keep our 5' and 3' ends properly oriented.
Original Sequence 5'ATGCAGGGGAAACATGATTCAGGAC 3'
Complement 3'TACGTCCCCTTTGTACTAAGTCCTG 5'
(Pairs with Original Sequence, antiparallel)
Reverse Complement 5'GTCCTGAATCATGTTTCCCCTGCAT 3'
(Complement sequence written 5' to 3')
A group of degenerate oligonucleotides contain related sequences with differences at specific locations. These are used simultaneously in the hope that one of the sequences of the oligonucleotides will be perfectly complementary to a target DNA sequence.
One common use of degenerate oligonucleotides is when the amino acid sequence of a protein is known. One can reverse translate this sequence to determine all of the possible nucleotide sequences that could encode that amino acid sequence. A set of degenerate oligonucleotides would then be produced matching those DNA sequences.
Also keep in mind that most oligonucleotide synthesis reactions are only 98% efficient. This means that each time a base is added, only 98% of the oligos will receive the base. This is not often critical with shorter oligos, but as length increases, so does the probability that a primer will be missing a base.
Oligonucleotide length / Percent with correct sequence
10 bases / (0.98)pwr10 = 81.7%
20 bases / (0.98)pwr20 = 66.7%
30 bases / (0.98)pwr30 = 54.6%
40 bases / (0.98)pwr40 = 44.6%