Improvement Of Pyrosequencing To Allow Multiplex Snp Typing In A Pyrogram

A multiplex single-nucleotide polymorphism (SNP) typing platform using "bioluminometric assay coupled with terminator (2', 3'-dideoxynucleoside triphosphates [ddNTPs]) incorporation" (named "BATI" for short) was developed. All of the reactions are carried out in a single reaction chamber containing target DNAs, DNA polymerase, reagents necessary for converting PPi into ATP, and reagents for luciferase reaction. Each of the four ddNTPs is dispensed into the reaction chamber in turn. PPi is released by a nucleotide incorporation reaction and is used to produce ATP when the ddNTP dispensed is complementary to the base in a template. The ATP is used in a luciferase reaction to release visible light. Only 1 nt is incorporated into a template at a time because ddNTPs do not have a 3' hydroxyl group. This feature greatly simplifies a sequencing spectrum. The luminescence is proportional to the amount of template incorporated. Only one peak appears in the spectrum of a homozygote sample, and two peaks at the same intensity appear for a heterozygote sample. In comparison with pyrosequencing using dNTP, the spectrum obtained by BATI is very simple, and it is very easy to determine SNPs accurately from it. As only one base is extended at a time and the extension signals are quantitative, the observed spectrum pattern is uniquely determined even for a sample containing multiplex SNPs. We have successfully used BATI to type various samples containing plural target sequence areas. The measurements can be carried out with an inexpensive and small luminometer using a photodiode array as the detector. It takes only a few minutes to determine multiplex SNPs. These results indicate that this novel multiplexed approach can significantly decrease the cost of SNP typing and increase the typing throughput with an inexpensive and small luminometer.