Hum Genet (1992) 89 : 361
9 Springer-Verlag1992
A polymorphic PstI site in intron 2 of the human apolipoprotein C-II gene detected by polymerase chain reaction Bernice R. Zysow, Clive R. Pullinger, and John P. Kane Cardiovascular Research Institute, Box 0130, University of California at San Francisco, San Francisco, CA 94131, USA Received October 17, 1991 / Revised November 15, 1991
Summary. A previously unrecorded polymorphism of the human apolipoprotein C-II gene, with results in the loss of a PstI site is described. Description. Primers (5' C C G A G T C A C A C A G A G C A G G A T C T C A G T C 3' and 5' A A G A A T T C A G G A -
C T A G C T A G A G A G T T G G G A G G A G G 3') in intron 1 and exon 4, respectively, were used to amplify a 1-kb genomic D N A fragment of the human apolipoprotein CII gene. One pg of genomic D N A , 50 ng of each primer, 200 ~tM of each dNTP, 25 m M Tris-HC1 (pH. 9.5), 50 m M KC1, 10 m M Mg2C1, 1 mg/ml BSA and 1 unit of Hot Tub polymerase (Amersham) were mixed in a 100-pl volume. Amplification conditions were 30 cycles of three steps (95~ for 15 s, 60~ for 15 s, and 72~ for 2.5 min). Polymorphism. The polymorphism occurs in the PstI site at nucleotide 2875 (Fojo et al. 1987), which is next to the junction of intron 2 and exon 3. When this PstI site is present on both alleles, digestion of the polymerase chain reaction (PCR) product with PstI results in fragments of 36 bp, 302 bp, and 675 bp. In the absence of this site from one allele, an additional fragment of 711 bp is seen (Fig. 1). A fragment of the PCR product obtained from two individuals displaying the polymorphism (lanes 1 and 5, Fig. 1) was subcloned into M13. Single-stranded D N A was prepared from a number of clones and sequencing was performed by the Sanger dideoxynucleotide chain termination method, using the Sequenase kit (USB). D N A sequence from these two individuals reveals that each is heterozygous for the same single nucleotide substitution of G-to-C at position 2877. Representative sequence ladders from one individual are shown in Fig. 2. Frequency. R a n d o m sample of 100 unrelated individuals: PstI site absent in 3% (1 Caucasian, and 2 African
Fig. 1. PstI digestion of PCR products showing a polymorphic PstI site in the human apoC-II gene. Lanes 1, 3 and 5 show three individuals heterozygous for the polymorphism, while individuals in lanes 2 and 4 are homozygous for the common allele
Fig. 2A, B. Sequencing gels showing the common allele (A), with the PstI site boxed, and the rare allele (B). The G-to-C substitution causing the loss of the PstI site is marked with an asterisk al. 1987). The 711-bp fragment from one individual is a doublet (lane 1, Fig. 1). The uppermost band is thought to be heteroduplex D N A due to a polymorphism (determined by sequencing) in the 37-nucleotide tandem repeat mini-satellite region in intron 3.
Americans). Chromosomal location. 19cen-19q13.2. Mendelian inheritance. Dominant in one two-generation
family studied (a total of i1 individuals). Comments. Apolipoprotein C-II was previously found not to be polymorphic for PstI using Southern blot analysis of genomic D N A (Frossard et al. 1986; Korneluk et Offprint requests to: B. R. Zysow
References Fojo SS, Law SW, Brewer HB Jr (1987) The human preproapolipoprotein C-II gene: complete nucleic acid sequence and genomic organization. FEBS Lett 213 : 221-226 Frossard PM, Coleman RT, Assman G (1986) Ncol RFLP at the human apolipoprotein CII gene locus. Nucleic Acids Res 14: 5120 Korneluk RG, MacLeod HL, Leblond SC, Monteith NL, Baralle FE, Hunter AGW (1987) Avail RFLP at the human apolipoprotein CII (APO CII) gene locus. Nucleic Acids Res 15 : 6769
9 Springer-Verlag1992
A polymorphic PstI site in intron 2 of the human apolipoprotein C-II gene detected by polymerase chain reaction Bernice R. Zysow, Clive R. Pullinger, and John P. Kane Cardiovascular Research Institute, Box 0130, University of California at San Francisco, San Francisco, CA 94131, USA Received October 17, 1991 / Revised November 15, 1991
Summary. A previously unrecorded polymorphism of the human apolipoprotein C-II gene, with results in the loss of a PstI site is described. Description. Primers (5' C C G A G T C A C A C A G A G C A G G A T C T C A G T C 3' and 5' A A G A A T T C A G G A -
C T A G C T A G A G A G T T G G G A G G A G G 3') in intron 1 and exon 4, respectively, were used to amplify a 1-kb genomic D N A fragment of the human apolipoprotein CII gene. One pg of genomic D N A , 50 ng of each primer, 200 ~tM of each dNTP, 25 m M Tris-HC1 (pH. 9.5), 50 m M KC1, 10 m M Mg2C1, 1 mg/ml BSA and 1 unit of Hot Tub polymerase (Amersham) were mixed in a 100-pl volume. Amplification conditions were 30 cycles of three steps (95~ for 15 s, 60~ for 15 s, and 72~ for 2.5 min). Polymorphism. The polymorphism occurs in the PstI site at nucleotide 2875 (Fojo et al. 1987), which is next to the junction of intron 2 and exon 3. When this PstI site is present on both alleles, digestion of the polymerase chain reaction (PCR) product with PstI results in fragments of 36 bp, 302 bp, and 675 bp. In the absence of this site from one allele, an additional fragment of 711 bp is seen (Fig. 1). A fragment of the PCR product obtained from two individuals displaying the polymorphism (lanes 1 and 5, Fig. 1) was subcloned into M13. Single-stranded D N A was prepared from a number of clones and sequencing was performed by the Sanger dideoxynucleotide chain termination method, using the Sequenase kit (USB). D N A sequence from these two individuals reveals that each is heterozygous for the same single nucleotide substitution of G-to-C at position 2877. Representative sequence ladders from one individual are shown in Fig. 2. Frequency. R a n d o m sample of 100 unrelated individuals: PstI site absent in 3% (1 Caucasian, and 2 African
Fig. 1. PstI digestion of PCR products showing a polymorphic PstI site in the human apoC-II gene. Lanes 1, 3 and 5 show three individuals heterozygous for the polymorphism, while individuals in lanes 2 and 4 are homozygous for the common allele
Fig. 2A, B. Sequencing gels showing the common allele (A), with the PstI site boxed, and the rare allele (B). The G-to-C substitution causing the loss of the PstI site is marked with an asterisk al. 1987). The 711-bp fragment from one individual is a doublet (lane 1, Fig. 1). The uppermost band is thought to be heteroduplex D N A due to a polymorphism (determined by sequencing) in the 37-nucleotide tandem repeat mini-satellite region in intron 3.
Americans). Chromosomal location. 19cen-19q13.2. Mendelian inheritance. Dominant in one two-generation
family studied (a total of i1 individuals). Comments. Apolipoprotein C-II was previously found not to be polymorphic for PstI using Southern blot analysis of genomic D N A (Frossard et al. 1986; Korneluk et Offprint requests to: B. R. Zysow
References Fojo SS, Law SW, Brewer HB Jr (1987) The human preproapolipoprotein C-II gene: complete nucleic acid sequence and genomic organization. FEBS Lett 213 : 221-226 Frossard PM, Coleman RT, Assman G (1986) Ncol RFLP at the human apolipoprotein CII gene locus. Nucleic Acids Res 14: 5120 Korneluk RG, MacLeod HL, Leblond SC, Monteith NL, Baralle FE, Hunter AGW (1987) Avail RFLP at the human apolipoprotein CII (APO CII) gene locus. Nucleic Acids Res 15 : 6769
