Set I and II of Fluorescent Primer Information
(Made in February and September, 1996)
Chr.Locus relative dye l_Pr lab size Mg No. annealin ref marker
location (mM) allel temp quality
1 Sw64 23.5 T A G 135-152 1.5 6 58 -16 ***/G
1 CGA 52.3 H B G 250-320 1.5 12 55 -4 ***/G
1 Sw781 55.8 H B G 123-198 1.5 8 55 -16 **/G
1 S0313 78.7 H B G 146-176 1.5 11 55 -2 ***/G
1 S0113 80.5 T A G 128-158 1.5 9 60 -17 ***/G
1 S0155 93.9 F A M 150-166 1.5 6 55 -4 ***/M
1 S0112 121.3 T B M/G 140-165 1.5 7 55 -17 ***/G
2 Sw256 19.2 F A M 92-118 1.5 7 62 -16 **/M
2 S0141 31.2 H A G 208-234 1.5 9 55 -18 **/G
2 Sw240 42.0 T A M/G 96-115 1.5 8 55 -16 ***/G
2 Sw395 66.1 F B G 143-161 1.5 8 55 -16 **/G
2 S0226 68.0 F B M 181-205 4 8 55 -14 ***/M
2 S0010 77.9 H A G 102-124 1.5 9 55 -7 ***/G
2 S0378 116.0 H A M 91-115 3 8 55 -13 ***/M
3 Sw72 17.8 F A M/G 100-116 1.5 5 58 -16 ***/M
3 S0206 42.3 F B M 175-205 4 7 60 -15 ***/M
3 Sw902 58.4 F A G 190-214 1.5 8 55 -16 ***/G
3 S0164 60.5 T A G 183-309 1.5 19 55 -4 ***/G
3 S0216 86.1 T A M/G 123-216 4 7 55 -15 ***/M
3 S0002 102.2 H A M 190-216 1.5 5 62 -7 ***/M
4 S0227 4.1 H A M/G 231-256 4 12 55 -14 **/M
4 S0301 27.1 F A G 252-268 1.5 7 55 -8 ***/G
4 S0001 41.8 F A G 177-200 1.5 6 55 -7 ***/G
4 S0214 79.3 T A M/G 124-160 1.5 8 55 -15 ***/G
4 S0097 120.0 T A M/G 181-246 1.5 13 58 -5 **/M
5 Swr453 57.9 H B M/G 172-190 1.5 4 55 -16 ***/G
5 S0005 88.2 T A M/G 205-248 1.5 11 58 -7 ***/M
5 IGF1 118.7 F A M/G 197-209 1.5 7 58 -10 ***/M
5 Sw967 145.9 H A M 95-114 1.5 7 58 -16 **/M
6 Sw1057 47.1 H A M 158-190 1.5 7 58 -16 **/M
6 S0087 62.8 T A M 165-213 1.5 8 58 -5 ***/M
6 Sw122 83.3 F B G 110-122 1.5 10 55 -16 ***/G
6 S0220 97.0 F A G 143-158 1.5 5 60 -14 ***/G
6 S0003 102.0 H A G 131-167 1.5 8 55 -7 ***/G
6 S0228 105.2 T A M 222-249 4 11 55 -14 **/M
7 S0025 3.7 F A G 104-121 1.5 2 55 -3 ***/G
7 S0064 30.2 F A G 148-180 1.5 7 55 -7 ***/G
7 S0102 70.1 T A M/G 120-141 1.5 7 55 -4 ***/M
7 Sw175 81.5 F A G 84-138 1.5 10 55 -16 ***/G
7 S0066 82.8 H A G 135-198 1.5 3 55 -7 ***/G
7 Sw632 104.4 T A M 159-180 1.5 6 58 -16 ***/M
7 S0101 134.9 H A M 197-216 1.5 6 60 -4 ***/M
8 Sw905 20.8 F B G 125-151 1.5 6 55 -16 ***/G
8 S0017 60.4 H A G 158-176 1.5 6 55 -3 ***/G
8 S0086 62.2 F A G 195-238 1.5 10 55 -5 **/G
8 S0225 82.8 H A M/G 170-196 4 9 55 -14 ***/M
8 Sw61 112.3 H A G 236-266 1.5 10 55 -16 ***/G
8 OPN(SPP 120.2 H B G 140-167 1.5 n.d. 60 -4 **/G
8 S0178 127.7 T A M 110-124 1.5 4 58 -4 ***/M
9 Sw911 36.8 T A M/G 153-177 1.5 7 55 -16 ***/M
9 Sw539 79.0 F A G 148-164 1.5 4 55 -16 ***/G
9 Sw174 122.9 F B M/G 123-131 1.5 5 55 -16 ***/G
10 Sw830 0.0 F A G 176-204 1.5 9 55 -16 ***/G
10 Sw249 17.3 H A G 130-156 1.5 8 55 -16 ***/G
10 Sw173 56.1 T A G 194-237 1.5 6 55 -16 ***/G
10 S0070 62.3 H A G 268-299 1.5 7 55 -7 ***/G
10 Sw920 86.3 F A G 142-159 1.5 4 55 -16 **/G
10 Sw951 96.0 H A M 125-133 1.5 7 58 -16 **/M
11 S0071 43.7 H A G 170-201 1.5 5 55 -7 ***/G
11 Sw435 53.3 T B G 148-187 1.5 6 55 -16 ***/G
11 S0230 56.4 F A G 282-328 1.5 10 55 -14 **/G
11 S0009 60.3 F A G 122-132 1.5 5 55 -7 ***/G
11 S0386 60.3 T A M 156-174 3 10 48 -12 **/M
12 S0143 6.6 T A G 154-164 1.5 4 55 -18 ***/G
12 Sw874 64.7 H B M/G 191-219 1.5 8 55 -16 **/G
12 S0090 80.2 F A M/G 244-251 1.5 6 55 -5 ***/G
12 S0106 95.8 H B G 120-150 1.5 4 55 -6 ***/G
12 Sw605 108.3 F A G 109-134 1.5 5 55 -16 ***/G
13 S0219 1.6 H B M/G 161-178 4 2 55 -14 no star/M
13 Swr1008 53.0 H A - 201-255 1.5 11 62 -16 ***
13 S0068 62.2 T B G 211-260 1.5 10 55 -7 ***/G
13 Sw398 79.3 H A G 166-193 1.5 9 55 -16 ***/G
13 Sw1056 96.1 T A - 150-182 1.5 5 62 -16 ***
13 Sw769 117.5 F B G 106-139 1.5 7 55 -16 ***/G
13 S0215 121.2 H A M 135-169 4 9 55 -14 ***/M
14 Sw857 7.4 H A M/G 144-160 1.5 7 55 -16 ***/G
14 Sw295 35.7 T A G 109-139 1.5 8 55 -16 ***/G
14 Sw210 46.3 F A G 218-250 1.5 11 55 -16 ***/G
14 S0007 60.0 H B M/G 174-202 1.5 11 55 -7 ***/G
14 Sw2515 108.7 F A G 90-108 1.5 5 55 -1 **/G
15 S0355 13.8 F A M 243-277 3 15 55 -11 ***/M
15 Sw1111 39.8 T B M/G 165-181 1.5 6 55 -16 ***/G
15 Sw936 88.5 F A M 80-117 1.5 13 58 -16 ***/M
15 Sw906 89.3 T A G 150-188 1.5 7 55 -16 **/G
15 Sw1119 119.9 H A M 149-179 1.5 9 60 -16 ***/M
16 S0026 46.9 H A G 92-106 1.5 4 55 -3 **/G
16 S0061 92.6 H B M/G 259-275 1.5 7 55 -7 ***/G
17 Swr1004 17.8 F A G 146-169 1.5 8 60 -16 **/G
17 Sw24 23.3 T A M 96-121 1.5 8 58 -16 ***/M
17 S0296 32.0 F A G 158-182 1.5 5 55 -9 ***/G
17 Sw840 48.6 F A M 123-139 1.5 6 53 -16 ***/M
18 Sw1023 5.0 F A G 94-117 1.5 5 55 -16 ***/G
18 Sw787 31.6 F A G 150-166 1.5 5 55 -16 ***/G
X Sw980 119.9 F A G 114-134 1.5 9 55 -16 **/G
X Sw707 108.6 H A G 0-101 1.5 5 55 -16 ***/G
X S0218 114.3 T A M 164-184 2 8 55 -14 ***/M
Dye: T=tet, F=fam, H=hex
l_Pr: labeled primer,
Lab: G=Groenen, M=Milan.
Size: Allele sizes are from Milan/Groenen list and may not be similar to
published allele sizes.
M. Groenen's Protocol for PCR of fluorescent primers.
Polymerase chain reaction (PCR) amplifications were carried out in 12
ul reactions containing 25-50 ng genomic DNA, 1.5 mM MgCl2,
50 mM KCl, 10 mM Tris-HCl pH=8.3, 1 mM tetra-methylammoniumchloride (TMAC),
200 uM dNTP, 0.25 U Goldstar polymerase (Eurogentec) and 30 ng of each
primer, one of which was labeled with a fluorescent dye at the 5"
end. The amplifications were as follows: 5 min 95o C followed
by 35 cycles of 30 s 94 o C, 45 s 55-60 o C and 90
s at 72 o C, finally followed by an extension step of 10 minutes
at 72 o C. PCR amplification products for several markers were
combined and analyzed simultaneously on a 6% denaturing polyacrylamide
gel on an automatic sequencer (ABI, Perkin Elmer). Electrophoresis was
performed for 3 hours on 12 cm gels, and the results were analyzed using
the Genescan and Genotyper software (ABI, Perkin Elmer).
PCR Protocols and General Information about these primers. Drs. D. Milan and M. Groenen on occasion used primers that differed between the two labs. The difference was either in the primer sequence itself or in which primer was labeled with the fluorescent dye. In general, it is not important to label systematically either the forward or the reverse primer. It is important to label the same primer if one wants to compare data between labs as both strands don't have the same molecular weight and one may obtain different artifactual bands with the forward versus the reverse primer being labeled. Data shared and compared between the two labs (Milan and Groenen) for the following loci Sw453, S0007, S0112, S0114, Sw1111, Sw174, Sw874, Sw905 needs to be viewed with caution for this reason. Table 1 lists marker quality in the last column. This refers to the optimum conditions to be used with the primers supplied. The primers which were labeled by the manufacturer with Tet begin with the symbol "&", Hex labeled primers begin with the symbol "@" and Fam labeled primers begin with the letter "O". For instance, the labeled primer for CGA begins @GAACTTATG… because it is labeled with Hex.
Please note, TMAC alters annealing temperatures.
D. Milan's lab conditions used to amplify loci with fluorescent primers
Locus name = numbered protocol with PCR conditions used for amplification of that locus.
S0001 = 35 S0002 = 29 S0005 = 28 S0007 = 33 S0025 = 34 S0026 = 34
S0061 = 30 S0087 = 28 S0090 = 28 S0097 = 28 S0101 = 31 S0101 = 31
S0102 = 34 S0112 = 34 S0155 = 34 S0178 = 28 S0206 = 58 SW1119 = 31
SW174 = 28 SW24 = 28 SW240 = 28 SW249 = 28 SW256 = 31 SW539 = 31
SW605 = 28 SW632 = 28 SW72 = 28 SW840 = 33 SW857 = 28 SW874 = 28
SW874 = 28 SW905 = 31 SW911 = 31 SW936 = 28 SW951 = 28 SW967 = 28
SWR453 = 35 IGF1 = 28
Cond # Ta ng DNA UNIT TAQ/PCR N cycles Mg Cl2 (mM) Primer(uM) 1 55 50 GIBCO 0,5 25 4 0,5 2 55 50 GIBCO 0,5 25 4 0,5 28 58 50 GIBCO 0,5 30 1,5 0,5 29 62 50 GIBCO 0,5 30 1,5 0,5 30 55 50 GIBCO 0,5 30 1 0,5 31 60 50 GIBCO 0,5 30 1,5 0,5 32 65 50 GIBCO 0,5 30 1,5 0,5 33 53 50 GIBCO 0,5 30 1,5 0,5 34 55 50 GIBCO 0,5 30 1,5 0,5 35 50 50 GIBCO 0,5 30 1 0,5 38 55 50 GIBCO 0,5 30 3 0,5 58 55 50 GIBCO 0,5 30 4 0,5 63 48 50 GIBCO 0,5 30 3 0,5
Ta= annealing temperature
PCR protocol for markers Swr1008 and SW1056 tested at ISU SWR1008 and Sw1056 were multiplexed in 12 ul reactions containing 37.5 ng genomic DNA, 1 X PCR buffer (Promega), 1.5 mM MgCl2, 1 mM TMAC, 200 uM each dNTP, 0.3 U Taq polymerase (Promega) and 5 pmol each primer. The PCR profile included 5 min at 95oC followed by 35 cycles of 30 sec at 94oC, 45 sec at 62oC and 90 sec at 72oC, ending with an extension step of 10 min at 72oC. PCR products were analyzed on 6% polyacrylamide gels on an automatic sequencer (ABI) using the Genescan and Genotyper software (ABI). References listed are the initial published reports for each locus. The sizes listed in the table may be different from those published. In a few instances, the primer sequences are totally different from the published sequences resulting in PCR products in the 200-300 bp range.
References
- Alexander, L.J., D.L. Troyer, G.A. Rohrer, T.P.L. Smith, L.B. Schook, and C.W. Beattie. 1996. Physical assignments of 68 porcine cosmid and lambda clones containing polymorphic microsatellites. Mammalian Genome 7:368-372.
- Andersson Dear, D.V. and J.R. Miller. 1994. Isolation of dinucleotide repeats from a pig chromosome 1-specific DNA library. Mammalian Genome 5:649-651.
- Coppieters, W., A. van de Weghe, L. Peelman, A. van Zeveren, and Y. Bouquet. 1993. Characterization of porcine polymorphic microsatellite loci. Animal Genetics. 24:163-170.
- Ellegren, H., B. Chowdhary, M. Johansson, L. Marklund, M. Fredholm, I. Gustavsson, and L. Andersson. 1994. A primary linkage map of the porcine genome reveals a low rate of genetic recombination. Genetics. 137:1089-1100.
- Ellegren, H., J. M, B. P. Chowdhary, S. Marklund, D. Ruyter, L. Marklund, P. B. Nielsen, I. Edfors-Lijia, I. Gustavsson, R. K. Juneja, and L. Andersson. 1993. Assignment of 20 microsatellite markers to the porcine linkage map. Genomics. 16:431-439.
- Ellegren, H., B. Chowdhary, M. Johansson, and L. Andersson. 1994. Integrating the porcine physical and linkage map using cosmid-derived markers. Animal Genetics 25:155-164.
- Fredholm, M., A. Wintero, K. Christenssen, B. Kristensen, N. P. Brauer, W. Davies, and A. Archibald. 1993. Characterization of 24 porcine (dA-dC)n-9dT-dG)n microsatellites: genotyping of unrelated animals from four breeds and linkage studies. Mammalian Genome. 4:187-192.
- Hoyheim, B., A. Keiserud, and P.D. Thomsen. 1994. A highly polymorphic porcine dinucleotide repeat S0301 (BHT 12) at chromosome 4p15. Animal Genetics 25:432.
- Hoyheim, B., A. Keiserud, and P.D. Thomsen. 1995. A highly polymorphic porcine dinucleotide repeat S0296 (BHT 137) at chromosome 17q13. Animal Genetics 26:58.
- Kirkpatrick, B. W. 1992. Identification of a conserved microsatellite site in the porcine and bovine insulin-like growth factor-I gene 5' flank. Animal Genetics. 23:543-548.
- Milan, D., N. Woloszyn, M. Yerle, P. Le Roy, M. Bonnet, J. Riquet, Y. Lahbib-Mansais, J. C. Caritez, A. Robic, P. Sellier, J. M. Elsen, and J. Gellin. 1996. Accurate mapping of the "acid meat" RN gene on genetic and physical maps of pig Chromosome 15. Mammalian Genome. 7:47-51.
- Riquet, J., D. Milan, W. N, A. Schmitz, F. Pitel, G. Frelat, and J. Gellin. 1995. A linkage map with microsatellites isolated from swine flow-sorted chromosome 11. Mammalian Genome. 6:623-628.
- Robic in preparation.
- Robic, A., M. Dalen, N. Woloszyn, D. Milan, J. Riquet, and J. Gellin. 1994. Isolation of 28 new porcine microsatellites revealing polymorphism. Mammalian Genome. 5:580-583.
- Robic, A., J. L. Parrou, M. Yerle, A. Goureau, M. Dalen, D. Milan, and J. Gellin. 1995. Pig microsatellites isolated from cosmids revealing polymorphism and localized on chromosomes. Animal Genetics. 26:1-6.
- Rohrer, G. A., L. J. Alexander, J. W. Keele, T. P. Smith, and C. W. Beattie. 1994. A microsatellite linkage map of the porcine genome. Genetics. 136:231-245.
- Ruyter, D., A. J. M. Verstege, and J. J. van der Poel. 1994. Five porcine polymorphic microsatellite markers. Animal Genetics. 25:53.
- Wilke, K., M. Jung, Y. Chen, and H. Geldermann. 1994. Porcine (GT)n sequences: Structure and association with dispersed and tandem repeats. Genomics 21:63-70.
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