DOGSET:

Microsatellite Primer Guide

• Microsatellite Distribution
• Primer3 Design Parameters
• M13 Tailed Primers
• Reverse Tail
• Protocols
  • M13 Tailed Primer Protocol
  • PCR Conditions
  • Thermocycling Program for M13 3-Primer PCRs
  • Post PCR

Microsatellite Distribution

All microsatellite loci are perfect repeats (n > 12 for di-nucleotide repeats and n > 6 for tetra-).

Primer3 Design Parameters

All of the primers in DOGSET are designed using standardized parameters:

M13 Tailed Primers

M13 labeled sequences:

      FAM = TTT CCC AGT CAC GAC GTT G
      NED = TAA AAC GAC GGC CAG TGC
      VIC = GCG GAT AAC AAT TTC ACA CAG G

Identical sequences appended to the 5' end of the locus-specific forward primers. For a description of the M13 tailed primer protocol please see: M13 Tailed Primer Protocol.

Reverse Tail

The following sequence can be appended to the 5' end of the reverse primer:

+A/-A Tail = GTTTCTT

This sequence serves to drive the non-templated addition by Taq of a base (usually +A) onto the 3' end of the labeled strand. This allows for easier allele scoring (by eliminating the -A peak).

M13 Tailed Primer Protocol

Primer Mixes

Labeled primer mix stock

• 52 µls of 500 uM M13, dye-labeled oligos (Applied Biosystems, see also M13 Tailed Primers) added to 50 mls of 10 mM Tris buffer
• each of the three labeled M13 primers at 0.52 uM in primer mix stock

Add the following to 2990 uls of M13 primer mix stock:

• 7.7 µls of each 100 uM Forward (usually a 40-mer; contains the M13 leader sequence) x 3 (multiplex 3-deep)
• 77 µls of each 100 uM Reverse (usually a 26-mer; contains the +A leader sequence) x 3 (multiplex 3-deep)
• plus 1016 µls of 10 mM Tris to bring to volume = 4260 µls of reagent; enough for 8 microtiter plates of PCR

Forward/ M13 /Reverse/ are in final ratio of 0.1x/0.2x/1.0x

X = 1.8 uM

PCR Conditions

DNA - 2 µls of cheek swab extract (standard extraction protocol) added to each well and allowed to desiccate (stable for at least several months at room temperature).

Alternatively, 50 ng pure DNA (extracted from tissue or blood) is used.

Primer mix (5 µls) is added to each well and covered with Chill-Out® (Bio-Rad); allows for easy hot-start setup.

PCRs for genotyping performed in 17-µl reactions

Final PCR concentrations:

• 2.5 mM MgCl₂ (AbGene)
• 1x buffer (AbGene)
• 200 µM dNTP (GenScript Corp)
• 1 unit of Taq DNA Polymerase (AbGene)

Cocktail per 96 well plate:

Hot-start: DNA with primer mix (5 µls) are brought to 93°C (covered with Chill-Out®). 12 µls of cocktail mix is then added to each well for final reaction volume of 17 µls.

Thermocycling Program for M13 3-Primer PCRs

1. 93° for 3 min
2. 93° for 20 sec
3. 7 cycles @ 65° for 30 sec
4. 72° for 2 min
5. 93° for 20 sec
6. 5 cycles @ 58° for 30 sec
7. 72° for 2 min
8. 93° for 20 sec
9. 25 cycles @ 55° for 30 sec
10. 72° for 2 min
11. 72° for 20 min

Post PCR

• 0.4 µl of PCR product is mixed with 2 µl of ABI GeneScan 500 LIZ fluorescent ladder
• Denatured for 3 min at 95°C; maintained at 5°C until loading.
• 1.5 µl aliquots loaded onto and analyzed with ABI 3730 capillary instrument.
• Genotypes scored with ABI GeneMapper software V4.0.
• If desirable, GM "binset" definitions available upon request (precision on capillary instruments may afford directly transferring these data).