Primer Design (go back)
The three relevant types of primers used in polony reactions are (1) an amplification primer with a 5'-acrydite modification, (2) an unmodified amplification primer, and (3) a sequencing primer.
With respect to amplification primers, the following is excerpted from the Supplementary Information of "Digital Genotyping and Haplotyping with Polymerase Colonies" (PNAS 2003).
"All primers used to perform the polony amplification reactions were designed by using PRIMER 3 software (3). We found it was necessary to set the following parameters to obtain good results: PRIMER_OPT_SIZE = 25, PRIMER_MIN_SIZE = 19, PRIMER_MAX_SIZE = 30, PRIMER_OPT_TM = 70, PRIMER_MIN_TM = 64, PRIMER_MAX_TM = 73, PRIMER_MAX_DIFF_TM = 5, PRIMER_MIN_GC = 45, PRIMER_MAX_GC = 80. For some experiments, the parameter PRIMER_PRODUCT_SIZE_RANGE = 90-100 was used. All other parameters were set to default values."
Link here to Primer3 software.
As far as sequencing primers, the position is usually determined by the base where you wish to begin sequencing. We generally choose a sequencing primer by extending 3' from this position until we have an oligonucleotide with a Tm of 68'C to 70'C.
Here are two sets of polony PCR amplification primers that have been particularly successful in our hands:
mCD44_a98F (5’ acrydite-modified): 5’-ACACGCTACAGCAAGAAGGGCGAGTAT-3’
mCD44_863R (unmodified): 5’-TGGTCTGGGGTCTCTGATGGTTCCT-3'
CPR (5’ acrydite-modified) 5’-GCCCGGTCTCGAGCGTCTGTTTA-3’
CPF (unmodified): 5’-GGGCGGAAGCTTGAAGGAGGTATT-3’