Determination of single-nucleotide polymorphisms by real-time pyrophosphate DNA sequencing

Abstract

The characterization of naturally occurring variations in the human genome has evoked an immense interest during recent years. Variations known as biallelic Single-Nucleotide Polymorphisms (SNPs) have become increasingly popular markers in molecular genetics because of their wide application both in evolutionary relationship studies and in the identification of susceptibility to common diseases. We have addressed the issue of SNP genotype determination by investigating variations within the Renin-Angiotensin-Aldosterone System (RAAS) using pyrosequencing, a real-time pyrophosphate detection technology. The method is based on indirect luminometric quantification of the pyrophosphate that is released as a result of nucleotide incorporation onto an amplified template. The technical platform employed comprises a highly automated sequencing instrument that allows the analysis of 96 samples within 10 to 20 minutes. In addition to each studied polymorphic position, 5-10 downstream bases were sequenced for acquisition of reference signals. Evaluation of pyrogram data was accomplished by comparison of peak heights, which are proportional to the number of incorporated nucleotides. Analysis of the pyrograms that resulted from alternate allelic configurations for each addressed SNP revealed a highly discriminating pattern. Homozygous samples produced clear-cut single base peaks in the expected position, whereas heterozygous counterparts were characterized by distinct half-height peaks representing both allelic positions. Whenever any of the allelic bases of an SNP formed a homopolymer with adjacent bases, the nonallelic signal was added to those of the SNP. This feature did not, however, influence SNP readability. Furthermore, the multibase reading capacity of the described system provides extensive flexibility in regard to the positioning of sequencing primers and allows the determination of several closely located SNPs in a single run.

Figure 1

Schematic outline of the procedure.

Figure 2

Identification of single nucleotide polymorphisms (SNPs) in biotin-labeled templates bound to streptavidin-coated paramagnetic beads (Dynabeads Streptavidin), by pyrosequencing. Primers were extended in the antisense direction. Sequencing primers and incorporated nucleotides are shown below each pyrogram series as an arrow and bold letters, respectively. Arbitrary luminescense units are shown on the ordinate axis. Each SNP is indicated by an arrowhead doublet. The two top diagrams show the respective homozygous test samples, whereas those below represent heterozygous counterparts. (A) A/T polymorphism, located in the human angiotensin I-converting enzyme promoter (A-240T). (B) C/T variation in the human angiotensin I-converting enzyme gene in exon 8 (T1237C). (C) Identical with (b), except that an allele-separating nucleotide dispensing scheme (within the biallelic variation) was applied.

Figure 2

Identification of single nucleotide polymorphisms (SNPs) in biotin-labeled templates bound to streptavidin-coated paramagnetic beads (Dynabeads Streptavidin), by pyrosequencing. Primers were extended in the antisense direction. Sequencing primers and incorporated nucleotides are shown below each pyrogram series as an arrow and bold letters, respectively. Arbitrary luminescense units are shown on the ordinate axis. Each SNP is indicated by an arrowhead doublet. The two top diagrams show the respective homozygous test samples, whereas those below represent heterozygous counterparts. (A) A/T polymorphism, located in the human angiotensin I-converting enzyme promoter (A-240T). (B) C/T variation in the human angiotensin I-converting enzyme gene in exon 8 (T1237C). (C) Identical with (b), except that an allele-separating nucleotide dispensing scheme (within the biallelic variation) was applied.

Figure 2

Identification of single nucleotide polymorphisms (SNPs) in biotin-labeled templates bound to streptavidin-coated paramagnetic beads (Dynabeads Streptavidin), by pyrosequencing. Primers were extended in the antisense direction. Sequencing primers and incorporated nucleotides are shown below each pyrogram series as an arrow and bold letters, respectively. Arbitrary luminescense units are shown on the ordinate axis. Each SNP is indicated by an arrowhead doublet. The two top diagrams show the respective homozygous test samples, whereas those below represent heterozygous counterparts. (A) A/T polymorphism, located in the human angiotensin I-converting enzyme promoter (A-240T). (B) C/T variation in the human angiotensin I-converting enzyme gene in exon 8 (T1237C). (C) Identical with (b), except that an allele-separating nucleotide dispensing scheme (within the biallelic variation) was applied.

Figure 3

Genotype assessment of a single SNP by pyrosequencing using two different primer oligonucleotides and heterozygous test samples. Below each pyrogram series, the respective sequencing primer (indicated by arrow) and incorporated bases (bold letters) are shown. Primers were extended in the sense direction. The ordinate represents luminescence (arbitrary units). Arrowhead doublets indicate the location of SNPs. The polymorphic position, addressed in both panels, corresponds to position A1166C in exon 5 of human angiotensin II type I receptor gene. (Upper panel) Extension of a sequencing primer, juxtaposed to the biallelic position. (Lower panel) Synthesis from a primer, located four bases upstream of the SNP.

Figure 4

Assessment of distinct SNPs on single fragments using real-time pyrophosphate detection. Arrowhead doublets indicate the location of SNPs. The schematic illustration below each pyrogram series shows the template, the primer location (arrow), and incorporated nucleotides (bold letters). (a) Variations in the human angiotensinogen promoter. Genotypes shown (at bottom) are: G/G, A/A and G/A (G-6A) and A/A, C/C and A/C (A-20C). (b) Polymorphisms, located in the promoter of human angiotensin II type I receptor gene. SNP configurations shown (top to bottom) correspond to G/G, C/C and G/C in position C-226G and A/A, C/C and A/C, in position A-227C.

Figure 4

Assessment of distinct SNPs on single fragments using real-time pyrophosphate detection. Arrowhead doublets indicate the location of SNPs. The schematic illustration below each pyrogram series shows the template, the primer location (arrow), and incorporated nucleotides (bold letters). (a) Variations in the human angiotensinogen promoter. Genotypes shown (at bottom) are: G/G, A/A and G/A (G-6A) and A/A, C/C and A/C (A-20C). (b) Polymorphisms, located in the promoter of human angiotensin II type I receptor gene. SNP configurations shown (top to bottom) correspond to G/G, C/C and G/C in position C-226G and A/A, C/C and A/C, in position A-227C.