Table 2

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

  1. 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.
  2. 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.
  3. 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.
  4. 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.
  5. 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.
  6. 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.
  7. 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.
  8. 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.
  9. 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.
  10. 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.
  11. 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.
  12. 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.
  13. Robic in preparation.
  14. 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.
  15. 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.
  16. 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.
  17. Ruyter, D., A. J. M. Verstege, and J. J. van der Poel. 1994. Five porcine polymorphic microsatellite markers. Animal Genetics. 25:53.
  18. 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.

Web Access Statistics © 1998-2019 NAGRP - U. S. Pig Genome Research Coordination Program.
Contact: The NAGRP Bioinformatics Project Team
Helpdesk