| | PCR amplifications. |
| | All the microsatellite markers described were studied using the |
| | following standard PCR conditions. These conditions represent a |
| | compromise which is probably remote from optimum for most primer |
| | pairs. |
| | PCR reactions are performed in a total volume of 50 micro liters, containing 80 |
| | ng of genomic DNA, 50 pmol of each primer, 0.125 mM dNTPs and 1 unit of |
| | Taq polymerase. 1X amplification buffer contains 10 mM Tris base pH9, |
| | 50 mM KCl, 1.5 mM MgCl2, 0.1% Triton X100 and 0.01% gelatin. The |
| | reactions are performed using a "hot-start" procedure: Taq polymerase |
| | is added only after a first denaturation step of 5 minutes at 96C degree. |
| | Amplification is carried out during 35 cycles of denaturation (94C degree for |
| | 40 sec) and annealing (55C degree for 30 sec). An elongation step (72C degree for 2 |
| | min) ends the process after the last annealing. |
| | This "hot-start" procedure permits an efficient primary denaturation |
| | step, and minimizes enzyme degradation. Moreover, addition of Taq |
| | polymerase at a high temperature, considerably reduces mispriming. |
| | Since the amplification products to be obtained are short (90 to 350 |
| | base pairs long) and the time interval to raise the temperature from 55 |
| | to 94C degree (obtained with a ramping rate of 1C degree/second) is long enough, |
| | completion of DNA elongation can be achieved without a step at 72C degree. |
| | This speeds up the overall amplification. |
| | Repeat Counts. |
| | Repeat counts were obtained on the reference sequence using Repeat Masker |
| | (http://repeatmasker.genome.washington.edu/). Repeat counts for the alleles |
| | are based on difference of the allele fragment size to reference fragment length. |
| | Ambiguous cases were submitted as named variations. |
