GENOMICS
9, 7x-77
(1991)
Development of Human Chromosome-Specific PCR Primers for Characterization of Somatic Cell Hybrids CATHY ABBOTT* AND SUE PovEyt *Department
of Genetics & Biometry, tMRC Human Biochemical Genetics Unit, University Wolfson House, 4 Stephenson Way, London NW1 ZHE, United Kjngdom Received
July
3, 1990
The human chromosome complement of somatic cell hybrids must be assessed each time the hybrids are grown in culture. We have developed a panel of human-specific oligonucleotide primers for genes that have been mapped to each of the autosomes and to the X chromosome. These primers enable the human chromosome content of hybrids to be assessed rapidly by PCR. The sequence of the primers is presented together with the appropriate conditions for human DNA-specific amplification for each pair. ,rl 1991 Academic Press. 1°C.
revised
September
1, 1990
have now developed a panel of human-specific oligonucleotide primers for each arm of 10 chromosomes and for one arm of the remaining 13 chromosomes (excluding the Y chromosome). In all cases described, the analysis is based on the presence or absence of the PCR product since this is the most rapid and simple analysis. However, it is also possible to design primers such that both the human gene and its rodent homolog are amplified but are subsequently distinguished from each other by, for example, differences in restriction sites. One such case is described.
INTRODUCTION
Somatic cell hybrids are an invaluable tool in human gene mapping studies. Genes can now be mapped by species-specific PCR in such hybrids (Abbott and Povey, 1990; Abbott et al., 1989). The human chromosome complement of hybrid cell lines must be assessedeach time the hybrids are grown in culture. In t.he past, this has been accomplished through a combination of karyotyping and isozyme analysis, usually by starch gel electrophoresis or isoelectric focusing. We have been developing a panel of oligonucleotide primers that enable us to characterize hybrids by PCR. PCR uses very little starting material, an advantage in itself and also because it reduces the need for a large-scale and therefore heterogeneous cell population. Primers for PCR must be capable of amplifying the human gene but not the equivalent rodent gene or of giving a different product for human and rodent. For this work, they were therefore chosen from noncoding regions of genes that have been sequenced and assigned to a particular human chromosome. Careful attention is paid to the conditions of PCR to ensure species-specificity. The presence or absence of the expected PCR product is checked by agarose gel electrophoresis and staining with ethidium bromide, and this is used to confirm the presence or absence of a particular human chromosome. We
College London,
METHOD
PCR was carried out using Taq polymerase from Anglian Biotec., Ltd, or Perkin-Elmer/Cetus or using Tet polymerase from the All-LJnion Research Institute of Influenza, Leningrad, USSR. Reactions were carried out. in a total volume of 100 ~1 with 10% DMSO, 1.5 mA4 deoxynucleotides, 50 pmol of each oligonucleotide, and 1.5 u Taq polymerase. The buffer used was 16.6 mA4 (NH,),SO,, 67 mM Tris-HCI, pH 8.8, 6.7 mM MgCl,, 10 mM @-mercaptoethanol, 6.7 pM EDTA, 170 pg/ml BSA. Cycling conditions were as described in Table 1, using a Hybaid Thermal Reactor. PCR product (10 ~1) was run on a 2% agarose gel and stained with ethidium bromide. RESULTS
Primer pairs have been synthesized and tested for human specificity for genes on chromosomes 1 (p + d, 2 (P + d, 3% 4 (P -t 91, 5 (P + d, 6 (P + q), 7 (P + d, 8 (P + d, 9 (P + d, 10 (P + q), 11 (P + d, 12 (P + d, lh, 1% 15% 16q, 17 (P + d, 18q, 19 (P + q),2Oq, 21q, 22q, and X (p + q) (Table 1). In all casesbut one, these primers were human-specific when appropriately stringent conditions were used (Fig. 1). The efficiency of the reaction was unaffected by the source of the thermostable polymerase used. In two cases, the
All
Copyright IC’ 1991 rights of reproduction
by Academic Press. Inc. in anv form reserved.
74
ABBOTT
AND
TABLE Sequences Chromosome
of Species-Specific
Ref.*
(;ene/region
1P
(31)
N(:F@/X
lq
(18)
Spectrin/intron
“p
(9)
T(:F~u/intron
“q
(“2)
Protein
:iq
ti)
linpuhlished
‘lP
(28)
HC/random
‘h
(13)
II,%/intron
5P
(4)
Complement
.5q
(8)
IL:‘,/intron
6P
(30)
Prolactin/intron
6q
(23)
Estrogen
7P
(10)
PGAM2/5’
7q
(ii)
CS7/random
XP
(26)
LHKH/:Y
8q
(iii)
CA:(/5’
9P
(17)
IFN
9q
(29)
Aldolase
1tJP
(211
Vimentin/intron
1oq
(24)
TUT/:1’
1lP
(27)
if-Glohin/intron
llq
(iv)
N-C:AM/5’
Up
(15)
CIs/:I’
1zq
(11)
Interf’eron-y/intron
13q
119)
Factor
1-h
(3)
cr,-Antitrypsin/intron
15q
(1%)
n-Actin
lfiq
(6)
Haptoglohin/3’
17P
(7)
~5315’ untrans
l7q
(1)
TCF2/intron
1%
(25)
Prealhumin/intron
19p
(34)
LDL
C/intron
CS/intron
receptor/intron untrans
unt,rans + intron
(u/3 untrans B/intron
untrans
untrans
untrans
VII/intron
tcardiac)/intron
recept,or/:i’
1 and Conditions Primer
untrans
untrans
Primers
POVEY
untrans
untrans
for Amplification
sequence
5’ GGAAGCATCCTGTGTGCTGATGCTG 5’ CTAACTTCTCCAAGGGAGGAGCCAG 5’ TAGAGAAGAAAGGCTGCCCAGTAGG 6’ CCTGGAACCAATGCCCCATGGATAA 5’ GATCTGAGCCCTGCATCTTTCCTCT 5’ GATCTCCAGGAGAACAGGGGATACT 5’ ATGTCATCATCCCACCCCATTCCAG 5’ AGCCTCTTGCTTAAGCACCTGGGGA .5’ GATTGGATCTCTTCCTTTTGATGAC 5’ CTGG(:GGA(;C:AAAACT(IAATAAAAT 5’ A(‘(‘TGGAT(“I’C(:GGCl”rC 5’ (;AACACAGAA’I’G(;GCTGC 5’ CTCACCTGCTTTTGTTCCCAGGGTA 5’ CCTATCCCTACCCCATCATAGTATC 5’ TAGATACATTGAGTCTCTCCTGATT 5’ (‘AGTCTACTACAATGAGAGAGATGG 5’ CATGGGTt:ATAACTACATCACCAGC 5’ GTACAGACATTCACAGCCACCCATA 5’ GGTTTCTGATACACTGGCCCGATAT 5’ GGAA(‘(:GATCATTAAGGACCTTCT(‘ 5’ C’I’(1T(:TCTCTGCGCAT’I’CA(: 5’ GAAG(‘(‘(‘A(:AC;AT(;CCTCAC 5’ (:(:TCCTA(:ACTCAACTCCG’I’GCCAC 5’ TCT(:(:C(‘T’rG’rGGAAGGTAC(IAGGC 5’ GAGA(;AAGC(:AAG(‘AATG 5’ TTAC:ACAC(:GG’rGCA’I’(;A 5’ AAATCCATTGGGCCAGAAGGAATGA 5’ G(:AA’I’AT(:‘I’GCAAC’rT(:GTGTAAGA 5’ ACTCTGCA(:CA(‘(:CA(;(:G(tAAGAGAGA 5’ A(:GTTA(:(;‘I’(:C7’TGTC(1AGTAAAAC 5’ GGAT’I’GAAAACTGGTTCAACATGGC 5’ TACTAGTGCCTGCACAGGTATACAC .5,’‘I’(‘AT’I’(:CT’r’(;C’TT’rCTCAAGCAGCAGGG 5’ CAATGCTTCTCCGTGTTGGAAAGTC 5’ AGTATCCC’ATCGCTCTTTCCCTGGC 5’ CATGAGCCTAGTCAGCAGAATGGTC 5’ A’1’(:(:(‘.I”1’G(:(‘(‘(‘(‘TTA’l”rCT(:’I’GAT 5’ ATT(:C’I’(‘T(:7’TC;(~~~‘r(‘TAACT(;CTC 5’ ACT(:G(:(‘ATGT(:GAGA(:AGAGAAGA 5’ TGTACCCTGT’I’ACTTCTCCCCTTCC 5’ TGGAAATCTCTTCCAAACATCGGAG 5’ AATTAGAACTTTGGAGAGGGATGGG 5 AGATACATCCCACCAGCCTCTCCAA 5’ GGAATAGTGCAAGAAAGGAGTGCCC 5’ (:CC:AT7”r(:(;C’I’AAT’I”I’TCC’rGTTGC 5’ (:AA1’AAA’I’(:4&1638.
CONCLUSION
The presence of any intact human chromosome can now be detected in somatic cell hybrids by speciesspecific PCR using this panel of human-specific primers. These primers should also be appropriate for use as sequence tagged sites. Although karyotyping is still necessary to characterize hybrids fully, this way of using PCR enables the initial characterization of hybrids to be carried out more quickly and efficiently than was possible by previous methods.
1:i.
discussions in the for the 4p and Yvonne Edwards Susan Tweedie a grant from the C.A.
early stage 79 primers, for the 7p for the 8q MRC lJ.K.
KOENIG;, M., MONACO, A.. AND KUNKEL. I,. (1988). plete sequence of dystrophin produces a rod-shaped t al protein. C’c~ll 53: ‘21 g-228.
15.
KIIS~IMOTO, H., HIHOSAWA, S., SALIER. J.. HA(:EN, KIIRA(:HI, K. (1988). Human genes for complement nents Clr and Cls in a close tail-to-tail arrangement. Nntl. /had. Sci. (‘.+‘A 85: 7307 7311.
16.
I,AVARON, 0.. ct (11. (1985). Architecture and anatomy of the chromosomal locus in human chromosome 2 1 encoding the (‘u/Zn superoxide dismutase. EMBO J. 4: 77 83. I,AWN, R.. ADEI,MAN, J., DULL, T.. GROSS, M., GOF,DI)EI,, I).. AND I:I,LRICH. A. (1981). D&A sequence of two closely linked human leukocyte interferon genes. Scienw 212: 1359-1162. I.INNF,NBA~H. A.. SPEICHER. I)., MAHCHESI, V., AND FORGET. Ii. I1986). Cloning oi’a portion of the human gene for human eryt hrocyte th-spectrin by using a synthetic gene fragment. I’rrx ,Vatl. Acad. Sci. ( ‘SA 83: 2397 ‘L401.
17.
REFERENCES 1.
ABBOTT, C., PIAGGIO, G., AMMENDOLA, R.. VEY. S., GOUNARI, F., DE SIMONE, V., (1990). Mapping of the gene TCF:! coding tion factor LFB3 to human chromosome chain reaction. Genomics 8: 165-167.
2.
ABBOTT, C., AND POVEY, S. (1990). The use of PCR in the mapping of human genes using somatic cell hybrids. In “Molecular Biology in Medicine” (C. Mathew. Ed.), Humana Press. in press.
3.
ABBOTT, C., POVEY, S., VIVIAN, (1988). PCR as a rapid screening Trends Genet. 4: 325.
4.
ABBOTT, C.. WEST, 1~. F., POVEY, S., JEREMIAH, S., M~JRAD, Z., DISCIPIO, R., AND FEY, G. (1989). The gene for human complement component C9 mapped to chromosome 5 hy PCR. Gcvxomics 4: 606-609.
51
6.
7.
SOLOMON, E., PoAND CORTESE, R. for the transcrip17 hy polymerase
N., AND LOVEI~L-BADGE. R. method for transgenic mice.
ANSON, D.. CHOO, K., REES, D., GIANELLI, F., GOULD, K., HUDDLESTON, J., AND BROWNLEE. G. (1984). The gene structure of human anti-haemophilic factor IX. EMBOJ. 3: 105% 1060. BENSI, G., RAUGEI, G., KLEFENZ, H., AND CORTESE, R. (1985). Structure and expression of the human haptoglobin locus. EMBO J. 4: 119-126. BIENZ-TADMOR, B., ZAKUT-HOURI, R., LIBRESCO, S., GIVOL, D., AND OREN, M. (1985). The 5’ region of the p53 gene: Evolutionary conservation and evidence for a negative regulatory element. EMBO J 4: 3209-321X.
of the human 859-863.
14.
ACKNOWLEDGMENTS We thank John McTigue for useful of this work. We thank Iain McIntosh Dallas Swallow for the 3q primers, primers, and Yvonne Edwards and primers. This work was supported by Human Genome Mapping Project to
R.. ROBERTS, A., WINKLEH. M.. CHEN, E., AND D. ( 1984). Human transforming growth factor-cl: structure and expression in E. co/i. c’ell 38: %87-
1X.
The comcytoskeleF.. AND compohoc.
19.
O’HAKA, I’., (IRAN?‘, F., HALUERMAN, H., (FRAY. C.. LNSLEY. M.. HAGEN, F.. AND MURRAY. M. (1987). Nwleotide sequence of the gene coding for human factor VII. a vitamin K-dependent protein participating in blood c~~agulation. Prclc. Nat/. Acnd. Sci. 1 rS.4 84: 5158~516”.
“0.
PAIK, Y.. (‘HAN(:, I)., REAKDON, (‘., DAVIES, G.. MAHLEY, R.. AND TAYLOR, .J. (198.5). Nucleotide sequence and structure of the human apolipoprotein E gene. F’roc. Natl. Acad. Sci. C:SA 82: :>41S-3449.
21.
PERRF,AU. .I., LII~IENBAUM, A., VASSEUR, M., AND PAIUN, D. (19881. Nucleotide sequence of the human vimentin gene and regulation 01’ its transcription in tissues and cultured cells. (;rv,e 62: 7- 16.
2”.
I’LU~ZKY, .I., HOSKINS, .J.. LONG, G.. ANI) CRABTREE, (1986). Evolution and organization of the human protein gene. I’roc. Natl. Acnd. Sci. USA 83: 546XiSO.
“3.
PON~LIKITMON~KOL, M.. GREEN, S., ANL) (:HAMBON, I’. (1988). Genomic organization of the human oestrogen receptor gene. EMBO J. 7: 3385~3388.
2-L.
HII.EY. I,., MORROW, .J., DANTON. M., AND (COLEMAN, M. ( 1988). Human terminal deoxyribonucleotidyltransferase: Molecular cloning and structural analysis of the gene and 5’ flanking region. Pruc. Natl. Acnd. Sci. C.‘S’A 85: 2489%249:1.
(:. (’
HUMAN
CHROMOSOME-SPECIFIC
25.
SASAKI, H., YOSHISKA, N., TAKAGI, (1985). Structure of the chromosomal bumin. Gene 37: 191-197.
26.
SEEBURG, P., AND ADEI,MAN, J. (1984). Characterization of cDNA for precursor of human luteinizing hormone releasing hormone. Nature (London) 311: 666-668. SPRITZ, R., et al. (1981). Base substitution in an intervening sequence of a p+-thalassaemic human globin gene. Proc. Natl. Acad. Sci. lI.SA 78: 2455-2459.
27.
Y., AND SAKAKI, Y. gene for serum preal-
28.
STAPLETON, P. (1988). Sequence analysis of three polymorphic regions in the human genome detected hy the G8 probe for RFLPs associated with Huntington’s disease. Nucleic Acids Res. 16: 27%.
29.
TOLAN, D. R., AND PENHOET, E. E. (1983). Characterization of the human aldolase B gene. Mol. Biol. Med. 3: 245-264.
30.
TRUONG,
A., D~Ez,
C., BELAYEW,
PCR
A., RENARD.
A., PICTET,
R.,
77
PRIMERS
BELL, G., AND MARTIAL, J. (1984). Isolation tion of the human prolactin gene. EMBO
and characterizaJ. 3: 429-437.
31.
ULLRICH, A., GRAY, A., BERMAN, C., AND DLJLL, T. (1983). Human o-nerve growth factor gene sequence highly homologous to that of mouse. Nature (London) 303: 821-825.
32.
WELLER, P., .JEFFREYS, A., WILSON, V., AND BLANCHETOT, A. (1984). Organization of the human myoglobin gene. EMROJ. 3: 439-446.
33.
WIGINTON, D., KAPI,AN, D., STATES, ,J., AKESON, A., PERME, C., BILYK, I., VAUGHN, A., LA’ITIER, D., AND H~J’ITON. J. (1986). Complete sequence and structure of the gene for human adenosine deaminase. Biochemistry 25: 8234-8’244.
34.
YAMAMOTO, T., DAVIS, C., BROWN, M., SCHNEIDER, W., CASEY, M., GOLDSTEIN, J., AND RUSSELL, D. (1984). The human I,DL receptor: A cysteine-rich protein with multiple Alu sequences in its mRNA. C’ell39: 27-38.
9, 7x-77
(1991)
Development of Human Chromosome-Specific PCR Primers for Characterization of Somatic Cell Hybrids CATHY ABBOTT* AND SUE PovEyt *Department
of Genetics & Biometry, tMRC Human Biochemical Genetics Unit, University Wolfson House, 4 Stephenson Way, London NW1 ZHE, United Kjngdom Received
July
3, 1990
The human chromosome complement of somatic cell hybrids must be assessed each time the hybrids are grown in culture. We have developed a panel of human-specific oligonucleotide primers for genes that have been mapped to each of the autosomes and to the X chromosome. These primers enable the human chromosome content of hybrids to be assessed rapidly by PCR. The sequence of the primers is presented together with the appropriate conditions for human DNA-specific amplification for each pair. ,rl 1991 Academic Press. 1°C.
revised
September
1, 1990
have now developed a panel of human-specific oligonucleotide primers for each arm of 10 chromosomes and for one arm of the remaining 13 chromosomes (excluding the Y chromosome). In all cases described, the analysis is based on the presence or absence of the PCR product since this is the most rapid and simple analysis. However, it is also possible to design primers such that both the human gene and its rodent homolog are amplified but are subsequently distinguished from each other by, for example, differences in restriction sites. One such case is described.
INTRODUCTION
Somatic cell hybrids are an invaluable tool in human gene mapping studies. Genes can now be mapped by species-specific PCR in such hybrids (Abbott and Povey, 1990; Abbott et al., 1989). The human chromosome complement of hybrid cell lines must be assessedeach time the hybrids are grown in culture. In t.he past, this has been accomplished through a combination of karyotyping and isozyme analysis, usually by starch gel electrophoresis or isoelectric focusing. We have been developing a panel of oligonucleotide primers that enable us to characterize hybrids by PCR. PCR uses very little starting material, an advantage in itself and also because it reduces the need for a large-scale and therefore heterogeneous cell population. Primers for PCR must be capable of amplifying the human gene but not the equivalent rodent gene or of giving a different product for human and rodent. For this work, they were therefore chosen from noncoding regions of genes that have been sequenced and assigned to a particular human chromosome. Careful attention is paid to the conditions of PCR to ensure species-specificity. The presence or absence of the expected PCR product is checked by agarose gel electrophoresis and staining with ethidium bromide, and this is used to confirm the presence or absence of a particular human chromosome. We
College London,
METHOD
PCR was carried out using Taq polymerase from Anglian Biotec., Ltd, or Perkin-Elmer/Cetus or using Tet polymerase from the All-LJnion Research Institute of Influenza, Leningrad, USSR. Reactions were carried out. in a total volume of 100 ~1 with 10% DMSO, 1.5 mA4 deoxynucleotides, 50 pmol of each oligonucleotide, and 1.5 u Taq polymerase. The buffer used was 16.6 mA4 (NH,),SO,, 67 mM Tris-HCI, pH 8.8, 6.7 mM MgCl,, 10 mM @-mercaptoethanol, 6.7 pM EDTA, 170 pg/ml BSA. Cycling conditions were as described in Table 1, using a Hybaid Thermal Reactor. PCR product (10 ~1) was run on a 2% agarose gel and stained with ethidium bromide. RESULTS
Primer pairs have been synthesized and tested for human specificity for genes on chromosomes 1 (p + d, 2 (P + d, 3% 4 (P -t 91, 5 (P + d, 6 (P + q), 7 (P + d, 8 (P + d, 9 (P + d, 10 (P + q), 11 (P + d, 12 (P + d, lh, 1% 15% 16q, 17 (P + d, 18q, 19 (P + q),2Oq, 21q, 22q, and X (p + q) (Table 1). In all casesbut one, these primers were human-specific when appropriately stringent conditions were used (Fig. 1). The efficiency of the reaction was unaffected by the source of the thermostable polymerase used. In two cases, the
All
Copyright IC’ 1991 rights of reproduction
by Academic Press. Inc. in anv form reserved.
74
ABBOTT
AND
TABLE Sequences Chromosome
of Species-Specific
Ref.*
(;ene/region
1P
(31)
N(:F@/X
lq
(18)
Spectrin/intron
“p
(9)
T(:F~u/intron
“q
(“2)
Protein
:iq
ti)
linpuhlished
‘lP
(28)
HC/random
‘h
(13)
II,%/intron
5P
(4)
Complement
.5q
(8)
IL:‘,/intron
6P
(30)
Prolactin/intron
6q
(23)
Estrogen
7P
(10)
PGAM2/5’
7q
(ii)
CS7/random
XP
(26)
LHKH/:Y
8q
(iii)
CA:(/5’
9P
(17)
IFN
9q
(29)
Aldolase
1tJP
(211
Vimentin/intron
1oq
(24)
TUT/:1’
1lP
(27)
if-Glohin/intron
llq
(iv)
N-C:AM/5’
Up
(15)
CIs/:I’
1zq
(11)
Interf’eron-y/intron
13q
119)
Factor
1-h
(3)
cr,-Antitrypsin/intron
15q
(1%)
n-Actin
lfiq
(6)
Haptoglohin/3’
17P
(7)
~5315’ untrans
l7q
(1)
TCF2/intron
1%
(25)
Prealhumin/intron
19p
(34)
LDL
C/intron
CS/intron
receptor/intron untrans
unt,rans + intron
(u/3 untrans B/intron
untrans
untrans
untrans
VII/intron
tcardiac)/intron
recept,or/:i’
1 and Conditions Primer
untrans
untrans
Primers
POVEY
untrans
untrans
for Amplification
sequence
5’ GGAAGCATCCTGTGTGCTGATGCTG 5’ CTAACTTCTCCAAGGGAGGAGCCAG 5’ TAGAGAAGAAAGGCTGCCCAGTAGG 6’ CCTGGAACCAATGCCCCATGGATAA 5’ GATCTGAGCCCTGCATCTTTCCTCT 5’ GATCTCCAGGAGAACAGGGGATACT 5’ ATGTCATCATCCCACCCCATTCCAG 5’ AGCCTCTTGCTTAAGCACCTGGGGA .5’ GATTGGATCTCTTCCTTTTGATGAC 5’ CTGG(:GGA(;C:AAAACT(IAATAAAAT 5’ A(‘(‘TGGAT(“I’C(:GGCl”rC 5’ (;AACACAGAA’I’G(;GCTGC 5’ CTCACCTGCTTTTGTTCCCAGGGTA 5’ CCTATCCCTACCCCATCATAGTATC 5’ TAGATACATTGAGTCTCTCCTGATT 5’ (‘AGTCTACTACAATGAGAGAGATGG 5’ CATGGGTt:ATAACTACATCACCAGC 5’ GTACAGACATTCACAGCCACCCATA 5’ GGTTTCTGATACACTGGCCCGATAT 5’ GGAA(‘(:GATCATTAAGGACCTTCT(‘ 5’ C’I’(1T(:TCTCTGCGCAT’I’CA(: 5’ GAAG(‘(‘(‘A(:AC;AT(;CCTCAC 5’ (:(:TCCTA(:ACTCAACTCCG’I’GCCAC 5’ TCT(:(:C(‘T’rG’rGGAAGGTAC(IAGGC 5’ GAGA(;AAGC(:AAG(‘AATG 5’ TTAC:ACAC(:GG’rGCA’I’(;A 5’ AAATCCATTGGGCCAGAAGGAATGA 5’ G(:AA’I’AT(:‘I’GCAAC’rT(:GTGTAAGA 5’ ACTCTGCA(:CA(‘(:CA(;(:G(tAAGAGAGA 5’ A(:GTTA(:(;‘I’(:C7’TGTC(1AGTAAAAC 5’ GGAT’I’GAAAACTGGTTCAACATGGC 5’ TACTAGTGCCTGCACAGGTATACAC .5,’‘I’(‘AT’I’(:CT’r’(;C’TT’rCTCAAGCAGCAGGG 5’ CAATGCTTCTCCGTGTTGGAAAGTC 5’ AGTATCCC’ATCGCTCTTTCCCTGGC 5’ CATGAGCCTAGTCAGCAGAATGGTC 5’ A’1’(:(:(‘.I”1’G(:(‘(‘(‘(‘TTA’l”rCT(:’I’GAT 5’ ATT(:C’I’(‘T(:7’TC;(~~~‘r(‘TAACT(;CTC 5’ ACT(:G(:(‘ATGT(:GAGA(:AGAGAAGA 5’ TGTACCCTGT’I’ACTTCTCCCCTTCC 5’ TGGAAATCTCTTCCAAACATCGGAG 5’ AATTAGAACTTTGGAGAGGGATGGG 5 AGATACATCCCACCAGCCTCTCCAA 5’ GGAATAGTGCAAGAAAGGAGTGCCC 5’ (:CC:AT7”r(:(;C’I’AAT’I”I’TCC’rGTTGC 5’ (:AA1’AAA’I’(:4&1638.
CONCLUSION
The presence of any intact human chromosome can now be detected in somatic cell hybrids by speciesspecific PCR using this panel of human-specific primers. These primers should also be appropriate for use as sequence tagged sites. Although karyotyping is still necessary to characterize hybrids fully, this way of using PCR enables the initial characterization of hybrids to be carried out more quickly and efficiently than was possible by previous methods.
1:i.
discussions in the for the 4p and Yvonne Edwards Susan Tweedie a grant from the C.A.
early stage 79 primers, for the 7p for the 8q MRC lJ.K.
KOENIG;, M., MONACO, A.. AND KUNKEL. I,. (1988). plete sequence of dystrophin produces a rod-shaped t al protein. C’c~ll 53: ‘21 g-228.
15.
KIIS~IMOTO, H., HIHOSAWA, S., SALIER. J.. HA(:EN, KIIRA(:HI, K. (1988). Human genes for complement nents Clr and Cls in a close tail-to-tail arrangement. Nntl. /had. Sci. (‘.+‘A 85: 7307 7311.
16.
I,AVARON, 0.. ct (11. (1985). Architecture and anatomy of the chromosomal locus in human chromosome 2 1 encoding the (‘u/Zn superoxide dismutase. EMBO J. 4: 77 83. I,AWN, R.. ADEI,MAN, J., DULL, T.. GROSS, M., GOF,DI)EI,, I).. AND I:I,LRICH. A. (1981). D&A sequence of two closely linked human leukocyte interferon genes. Scienw 212: 1359-1162. I.INNF,NBA~H. A.. SPEICHER. I)., MAHCHESI, V., AND FORGET. Ii. I1986). Cloning oi’a portion of the human gene for human eryt hrocyte th-spectrin by using a synthetic gene fragment. I’rrx ,Vatl. Acad. Sci. ( ‘SA 83: 2397 ‘L401.
17.
REFERENCES 1.
ABBOTT, C., PIAGGIO, G., AMMENDOLA, R.. VEY. S., GOUNARI, F., DE SIMONE, V., (1990). Mapping of the gene TCF:! coding tion factor LFB3 to human chromosome chain reaction. Genomics 8: 165-167.
2.
ABBOTT, C., AND POVEY, S. (1990). The use of PCR in the mapping of human genes using somatic cell hybrids. In “Molecular Biology in Medicine” (C. Mathew. Ed.), Humana Press. in press.
3.
ABBOTT, C., POVEY, S., VIVIAN, (1988). PCR as a rapid screening Trends Genet. 4: 325.
4.
ABBOTT, C.. WEST, 1~. F., POVEY, S., JEREMIAH, S., M~JRAD, Z., DISCIPIO, R., AND FEY, G. (1989). The gene for human complement component C9 mapped to chromosome 5 hy PCR. Gcvxomics 4: 606-609.
51
6.
7.
SOLOMON, E., PoAND CORTESE, R. for the transcrip17 hy polymerase
N., AND LOVEI~L-BADGE. R. method for transgenic mice.
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14.
ACKNOWLEDGMENTS We thank John McTigue for useful of this work. We thank Iain McIntosh Dallas Swallow for the 3q primers, primers, and Yvonne Edwards and primers. This work was supported by Human Genome Mapping Project to
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