Research paper
Analysis of multiple single nucleotide polymorphisms (SNP) on DNA traces from plasma and dried blood samples

https://doi.org/10.1016/j.jim.2007.01.015Get rights and content

Abstract

Reliable analysis of single nucleotide polymorphisms (SNPs) in DNA derived from samples containing low numbers of cells or from suboptimal sources can be difficult. A new procedure to characterize multiple SNPs in traces of DNA from plasma and old dried blood samples was developed. Six SNPs in the Mannose Binding Lectin 2 (MBL2) gene were chosen as targets for analysis.

DNA was extracted from plasma obtained from mothers (n = 49) and their neonates (n = 49) and from old dried blood samples (n = 204). Multiple Real-Time SNP analyses in the MBL2 gene were carried out on all samples. Because of very low DNA concentrations in most of the samples, a pre-amplification step was utilized.

It was possible to analyze all plasma samples (n = 98), including those with very low cell numbers (n = 21) and 93% of the old dried blood samples (n = 189). Results obtained from pre-amplified samples were in full agreement with neat samples. All possible SNP alleles were present in our population. The frequencies of the different alleles from both plasma and dried blood samples (n = 287) were in agreement with earlier studies of the Caucasian population.

In conclusion, amplification prior to Real-Time PCR SNP analysis is a convenient, cost effective and useful method to significantly improve the reliable SNP detection in specimens containing very low concentrations or poor quality DNA from suboptimal sources.

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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