TY - JOUR

T1 - Whole-genome amplified DNA from stored dried blood spots is reliable in high resolution melting curve and sequencing analysis

AU - Winkel, Bo G

AU - Hollegaard, Mads V

AU - Olesen, Morten S

AU - Svendsen, Jesper H

AU - Haunsø, Stig

AU - Hougaard, David M

AU - Tfelt-Hansen, Jacob

PY - 2011/2/9

Y1 - 2011/2/9

N2 - BACKGROUND: The use of dried blood spots (DBS) samples in genomic workup has been limited by the relative low amounts of genomic DNA (gDNA) they contain. It remains to be proven that whole genome amplified DNA (wgaDNA) from stored DBS samples, constitutes a reliable alternative to gDNA.We wanted to compare melting curves and sequencing results from wgaDNA derived from DBS samples with gDNA derived from whole blood.METHODS: gDNA was extracted from whole blood obtained from 10 patients with lone atrial fibrillation (mean age 22.3 years). From their newborn DBS samples, stored at -24°C, genomic DNA was extracted and whole-genome amplified in triplicates. Using high resolution melting curve analysis and direct sequencing in both wgaDNA and gDNA samples, all coding regions and adjacent intron regions of the genes SCN5A and KCNA5 were investigated.RESULTS: Altered melting curves was present in 85 of wgaDNA samples and 81 of gDNA samples. Sequence analysis identified a total of 31 variants in the 10 wgaDNA samples. The same 31 variants were found in the exact same pattern of samples in the gDNA group. There was no false positive or negative sequence variation in the wgaDNA group.CONCLUSIONS: The use of DNA amplified in triplicates from DBS samples is reliable and can be used both for high resolution curve melting analysis as well as direct sequence analysis. DBS samples therefore can serve as an alternative to whole blood in sequence analysis.

AB - BACKGROUND: The use of dried blood spots (DBS) samples in genomic workup has been limited by the relative low amounts of genomic DNA (gDNA) they contain. It remains to be proven that whole genome amplified DNA (wgaDNA) from stored DBS samples, constitutes a reliable alternative to gDNA.We wanted to compare melting curves and sequencing results from wgaDNA derived from DBS samples with gDNA derived from whole blood.METHODS: gDNA was extracted from whole blood obtained from 10 patients with lone atrial fibrillation (mean age 22.3 years). From their newborn DBS samples, stored at -24°C, genomic DNA was extracted and whole-genome amplified in triplicates. Using high resolution melting curve analysis and direct sequencing in both wgaDNA and gDNA samples, all coding regions and adjacent intron regions of the genes SCN5A and KCNA5 were investigated.RESULTS: Altered melting curves was present in 85 of wgaDNA samples and 81 of gDNA samples. Sequence analysis identified a total of 31 variants in the 10 wgaDNA samples. The same 31 variants were found in the exact same pattern of samples in the gDNA group. There was no false positive or negative sequence variation in the wgaDNA group.CONCLUSIONS: The use of DNA amplified in triplicates from DBS samples is reliable and can be used both for high resolution curve melting analysis as well as direct sequence analysis. DBS samples therefore can serve as an alternative to whole blood in sequence analysis.

KW - Adult

KW - Blood Preservation/methods

KW - Blood Specimen Collection/methods

KW - Genome

KW - Genome, Human/genetics

KW - Genomics/methods

KW - Hot Temperature

KW - Humans

KW - Infant, Newborn

KW - Kv1.5 Potassium Channel/genetics

KW - Muscle Proteins/genetics

KW - NAV1.5 Voltage-Gated Sodium Channel

KW - Nucleic Acid Amplification Techniques

KW - Phase Transition

KW - Sodium Channels/genetics

KW - Specimen Handling/methods

U2 - 10.1186/1471-2350-12-22

DO - 10.1186/1471-2350-12-22

M3 - Journal article

C2 - 21306642

VL - 12

SP - 22

JO - B M C Medical Genetics

JF - B M C Medical Genetics

SN - 1471-2350

ER -