67Molecular Inversion Probe Based Assays
A molecular inversion probe (MIP) is an unmodifi ed oligonucleotide (110–140
bases) that contains the following functional segments:
- Two sequences homologous to sites adjacent to a SNP of interest
- A unique tag sequence specifi c to each MIP that can be hybridized to an array
- Two sequences that anneal PCR primers common to all MIP probes
Using Lab-in-a-tube™ technology MIP enables multiplexed SNP scoring to lev-
els of 10,000 reactions in parallel. MIP genotyping is particularly effective when
genotyping >500 SNP’s with >100 samples.
Pyrosequencing
Pyrosequencing technology is based on DNA sequencing and is aimed at laborato-
ries performing medium to high-throughput screening of characterized SNPs.
Pyrosequencing is a sequencing-by-synthesis method in which a cascade of enzy-
matic reactions yields detectable light, which is proportional to incorporated nucle-
otides In contrast to some of the other methods discussed, pyrosequencing takes
into account the DNA sequence fl anking the SNP, which helps to reveal sequencing
artifacts and so reduces the number of false positive results which are generated.
Unlabeled sequencing primer is hybridized to the DNA target (PCR product) and a
single labeled ddNTP is added to the reaction mixture containing the enzymes DNA
polymerase, ATP sulfurylase, luciferase and apyrase in the presence of the sub-
strates adenosine 5′ phosphosulfate (APS) and luciferin. If ddNTP is incorporated
into the DNA strand, pyrophosphate (PPi) is released and reacts with APS to gener-
ate ATP in equimolar quantities to the amount of ddNTP introduced into the nascent
strand. ATP drives the conversion of luciferin to oxyluciferin, which generates light
as a by-product. The light is detected by a CCD camera and is registered as a single
peak on the pyrogram. Any remaining ATP and unincorporated ddNTP is upgraded
by the action of apyrase prior to addition of the second ddNTP. As the process con-
tinues, the DNA strand is extended and the nucleotide sequence is determined from
the relative height and number of signal peaks in the pyrogram.
Pyrosequencing enables genotyping of 96 samples within 10 min with an accu-
racy of >99 %. It can also be used to determine allele frequencies of known SNPs in
pooled populations of genomic DNA. Pyrosequencing technology offers a highly
automated, rapid, and accurate method for identifi cation of cytochrome P450
alleles, which is suitable for pharmacogenomic research, as well as for routine
assessment of patient genotypes. In pyrosequencing, a single set of PCR and
sequencing primers is used to coamplify and sequence a region in the CYP2D6 gene
and the equivalent region in the CYP2D8P pseudogene with relative quantifi cation
between these fragments. The use of an internal CYP2D8P control as well as gen-
eration of a sequence context insures robustness and facilitates validation of the
method.
SNP Genotyping