Research paperAnalysis of multiple single nucleotide polymorphisms (SNP) on DNA traces from plasma and dried blood samples
Introduction
Single nucleotide polymorphisms (SNPs) are the most frequent occurring manifestation of genetic variation in the human genome. Approximately one out of every 1000 nucleotides in the human genome is expected to be a SNP site, accounting for more than 90% of all differences between humans. SNPs contribute to the variation in human phenotypes, such as disease susceptibility, responses to drugs and environmental chemicals, and susceptibility to infection (Landegren et al., 1998, Twyman, 2004, Zhou et al., 2005). SNP identification and functional assessment is becoming an increasingly more important tool in molecular diagnostics and biology. Several different genotyping approaches are in rapid development, such as fluorescence homogenous assays, pyrosequencing, Real-Time PCR and mass spectrometry (Alderborn et al., 2000, Griffin and Smith, 2000, Livak, 1999).
At present, Real-Time PCR is the most used technology for detection of SNPs (Kwok, 2002). This assay requires only a small amount of purified DNA. In general, the amount of human DNA in clinical samples is more than sufficient. However, under some circumstances, e.g. when the sources of DNA are samples with very small cell numbers or older samples that have not been stored under optimal conditions for DNA preservation, the amount of DNA may be too low to yield reliable SNP results.
We evaluated a new procedure to characterize multiple SNPs in traces of DNA from plasma and old dried blood samples, using the MBL2 gene as proof of principle. This gene encodes the Mannose Binding Lectin 2, a C-type lectin that plays a role in the innate immune response to infections. MBL has a bouquet-like structure and it binds repeating oligosaccharides present in a wide variety of bacteria and other microbes. This is followed by a conformation change of Mannose Associated Serine Protease which results in the activation of the lectin pathway of the complement system (Kilpatrick, 2002). As a consequence, the microorganism is neutralized. MBL deficiency and low levels of serum MBL are strongly associated with the presence of six SNPs in the MBL2 gene (Madsen et al., 1995).
In this study, a new procedure that utilizes a pre-amplification step to improve the reliable identification of multiple SNPs in traces of DNA from plasma and old dried blood samples was validated using the MBL2 gene as target.
Section snippets
Blood samples
The samples consisted of plasma specimens collected from 49 mothers and their neonates, and 204, 3–5 year-old, blood spots on Guthrie cards from newborn children. All persons were of Dutch Caucasian ethnicity. The study was approved by the local ethics committee. Parents of eligible neonates were asked for permission (informed consent) to use the Guthrie card of their child.
DNA extraction
DNA was isolated from plasma samples with the QIAamp Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's
Control based genotype definition
To obtain all possible SNP controls, four different haplotypes were cloned in pGEM vectors: pLXPA, pHYPD, pLYPB and pLYQC. DNA derived from homozygous wild type samples was detected as a VIC fluorescence signal on the X-axis, whereas homozygous mutant samples were detected as a FAM fluorescence signal on the Y-axis. Heterozygous samples gave an intermediate fluorescence. For example, equal amounts of pLXPA and pHYPD represented a heterozygous H/L control.
SNP detection limit
For most of the alleles a lower
Discussion
Real-Time PCR is one of the most frequently used techniques for routine SNP genotyping. Usually, whole blood or WBC fractions are used as sources of DNA. Sometimes the only specimen available contains very small amounts of DNA or even only fragmented DNA. In such cases, SNP analysis may be unreliable. In this study we evaluated a new approach for typing multiple SNPs in one gene on very low amounts of DNA. As a model, we selected the MBL2 gene, which encodes the Mannose Binding Lectin 2
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