PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-25 (1224861)

Clipboard (0)
None

Related Articles

1.  Robustness of genome-wide scanning using archived dried blood spot samples as a DNA source 
BMC Genetics  2011;12:58.
Background
The search to identify disease-susceptible genes requires access to biological material from numerous well-characterized subjects. Archived residual dried blood spot (DBS) samples, also known as Guthrie cards, from national newborn screening programs may provide a DNA source for entire populations. Combined with clinical information from medical registries, DBS samples could provide a rich source for productive research. However, the amounts of DNA which can be extracted from these precious samples are minute and may be prohibitive for numerous genotypings. Previously, we demonstrated that DBS DNA can be whole-genome amplified and used for reliable genetic analysis on different platforms, including genome-wide scanning arrays. However, it remains unclear whether this approach is workable on a large sample scale. We examined the robustness of using DBS samples for whole-genome amplification following genome-wide scanning, using arrays from Illumina and Affymetrix.
Results
This study is based on 4,641 DBS samples from the Danish Newborn Screening Biobank, extracted for three separate genome-wide association studies. The amount of amplified DNA was significantly (P < 0.05) affected by the year of storage and storage conditions. Nine (0.2%) DBS samples failed whole-genome amplification. A total of 4,586 (98.8%) samples met our criterion of success of a genetic call-rate above 97%. The three studies used different arrays, with mean genotyping call-rates of 99.385% (Illumina Infinium Human610-Quad), 99.722% (Illumina Infinium HD HumanOmni1-Quad), and 99.206% (Affymetrix Axiom Genome-Wide CEU). We observed a concordance rate of 99.997% in the 38 methodological replications, and 99.999% in the 27 technical replications. Handling variables such as time of storage, storage conditions and type of filter paper were shown too significantly (P < 0.05) affect the genotype call-rates in some of the arrays, although the effect was minimal.
Conclusion
Our study indicates that archived DBS samples from the Danish Newborn Screening Biobank represent a reliable resource of DNA for whole-genome amplification and subsequent genome-wide association studies. With call-rates equivalent to high quality DNA samples, our results point to new opportunities for using the neonatal biobanks available worldwide in the hunt for genetic components of disease.
doi:10.1186/1471-2156-12-58
PMCID: PMC3142526  PMID: 21726430
2.  High Quality Genome-Wide Genotyping from Archived Dried Blood Spots without DNA Amplification 
PLoS ONE  2013;8(5):e64710.
Spots of blood are routinely collected from newborn babies onto filter paper called Guthrie cards and used to screen for metabolic and genetic disorders. The archived dried blood spots are an important and precious resource for genomic research. Whole genome amplification of dried blood spot DNA has been used to provide DNA for genome-wide SNP genotyping. Here we describe a 96 well format procedure to extract DNA from a portion of a dried blood spot that provides sufficient unamplified genomic DNA for genome-wide single nucleotide polymorphism (SNP) genotyping. We show that SNP genotyping of the unamplified DNA is more robust than genotyping amplified dried blood spot DNA, is comparable in cost, and can be done with thousands of samples. This procedure can be used for genome-wide association studies and other large-scale genomic analyses that require robust, high-accuracy genotyping of dried blood spot DNA.
doi:10.1371/journal.pone.0064710
PMCID: PMC3667813  PMID: 23737996
3.  Whole genome microarray analysis, from neonatal blood cards 
BMC Genetics  2009;10:38.
Background
Neonatal blood, obtained from a heel stick and stored dry on paper cards, has been the standard for birth defects screening for 50 years. Such dried blood samples are used, primarily, for analysis of small-molecule analytes. More recently, the DNA complement of such dried blood cards has been used for targeted genetic testing, such as for single nucleotide polymorphism in cystic fibrosis. Expansion of such testing to include polygenic traits, and perhaps whole genome scanning, has been discussed as a formal possibility. However, until now the amount of DNA that might be obtained from such dried blood cards has been limiting, due to inefficient DNA recovery technology.
Results
A new technology is employed for efficient DNA release from a standard neonatal blood card. Using standard Guthrie cards, stored an average of ten years post-collection, about 1/40th of the air-dried neonatal blood specimen (two 3 mm punches) was processed to obtain DNA that was sufficient in mass and quality for direct use in microarray-based whole genome scanning. Using that same DNA release technology, it is also shown that approximately 1/250th of the original purified DNA (about 1 ng) could be subjected to whole genome amplification, thus yielding an additional microgram of amplified DNA product. That amplified DNA product was then used in microarray analysis and yielded statistical concordance of 99% or greater to the primary, unamplified DNA sample.
Conclusion
Together, these data suggest that DNA obtained from less than 10% of a standard neonatal blood specimen, stored dry for several years on a Guthrie card, can support a program of genome-wide neonatal genetic testing.
doi:10.1186/1471-2156-10-38
PMCID: PMC2722673  PMID: 19624846
4.  Long-term storage limits PCR-based analyses of malaria parasites in archival dried blood spots 
Malaria Journal  2012;11:339.
Background
Blood samples collected in epidemiological and clinical investigations and then stored, often at room temperature, as blood spots dried on a filter paper have become one of the most popular source of material for further molecular analyses of malaria parasites. The dried blood spots are often archived so that they can be used for further retrospective investigations of parasite prevalence, or as new genetic markers come to the fore. However, the suitability of the template obtained from dried blood spots that have been stored for long periods for DNA amplification is not known.
Methods
DNA from 267 archived blood spots collected over a period of 12 years from persons with microscopically confirmed Plasmodium falciparum infection was purified by one of two methods, Chelex and Qiagen columns. These templates were subjected to highly sensitive nested PCR amplification targeting three parasite loci that differ in length and/or copy number.
Results
When a 1.6 kb fragment of the parasites’ small subunit ribosomal RNA was targeted (primary amplification), the efficiency of P. falciparum detection decreased in samples archived for more than six years, reaching very low levels for those stored for more than 10 years. Positive amplification was generally obtained more often with Qiagen-extracted templates. P. falciparum could be detected in 32 of the 40 negative Qiagen-extracted templates when a microsatellite of about 180 bp was targeted. The remaining eight samples gave a positive amplification when a small region of 238 bp of the higher copy number (20 to 200) mitochondrial genome was targeted.
Conclusions
The average length of DNA fragments that can be recovered from dried blood spots decreases with storage time. Recovery of the DNA is somewhat improved, especially in older samples, by the use of a commercial DNA purification column, but targets larger than 1.5 kb are unlikely to be present 10 years after the initial blood collection, when the average length of the DNA fragments present is likely to be around a few hundred bp. In conclusion, the utility of archived dried blood spots for molecular analyses decreases with storage time.
doi:10.1186/1475-2875-11-339
PMCID: PMC3507721  PMID: 23043522
Archival blood spots; Plasmodium falciparum; Polymerase chain reaction
5.  The use of DNA from archival dried blood spots with the Infinium HumanMethylation450 array 
BMC Biotechnology  2013;13:23.
Background
Dried blood (Guthrie card) spots provide an efficient way to collect and store blood specimens. DNA from this source has been utilised for a number of molecular analyses including genome-wide association studies, but only few studies have tested the feasibility of using it for epigenetic applications, particularly at a genome-wide level.
Results
In this study, we demonstrate the successful use of DNA isolated from archived dried blood spots for the Infinium HumanMethylation450 Beadchip, along with DNA from matched frozen buffy coats. We obtained high quality and reproducible genome-wide DNA methylation profiles using both sample types. We also report high correlations (r > 0.9907) between DNA obtained from matched dried blood spots and frozen buffy coats, sufficient to distinguish between unrelated individuals.
Conclusions
We, thus, demonstrate that DNA from archived dried blood spots is suitable for genome-wide DNA methylation profiling.
doi:10.1186/1472-6750-13-23
PMCID: PMC3610215  PMID: 23497093
DNA methylation; Epigenetics; Infinium HumanMethylation450; Dried blood spots; Guthrie cards; Genome-wide DNA methylation
6.  Isolation of human genomic DNA for genetic analysis from premature neonates: a comparison between newborn dried blood spots, whole blood and umbilical cord tissue 
BMC Genetics  2013;14:105.
Background
Genotyping requires biological sample collection that must be reliable, convenient and acceptable for patients and clinicians. Finding the most optimal procedure of sample collection for premature neonates who have a very limited blood volume is a particular challenge. The aim of the current study was to evaluate the use of umbilical cord (UC) tissue and newborn dried blood spot (DBS)-extracted genomic DNA (gDNA) as an alternative to venous blood-derived gDNA from premature neonates for molecular genetic analysis.
All samples were obtained from premature newborn infants between 24-32 weeks of gestation. Paired blood and UC samples were collected from 31 study participants. gDNA was extracted from ethylenediaminetetraacetic acid (EDTA) anticoagulant-treated blood samples (~500 μl) and newborn DBSs (n = 723) using QIAamp DNA Micro kit (Qiagen Ltd., Crawley, UK); and from UC using Qiagen DNAeasy Blood and Tissue kit (Qiagen Ltd., Crawley, UK). gDNA was quantified and purity confirmed by measuring the A260:A280 ratio. PCR amplification and pyrosequencing was carried out to determine suitability of the gDNA for molecular genetic analysis. Minor allele frequency of two unrelated single nucleotide polymorphisms (SNPs) was calculated using the entire cohort.
Results
Both whole blood samples and UC tissue provided good quality and yield of gDNA, which was considerably less from newborn DBS. The gDNA purity was also reduced after 3 years of storage of the newborn DBS. PCR amplification of three unrelated genes resulted in clear products in all whole blood and UC samples and 86%-100% of newborn DBS. Genotyping using pyrosequencing showed 100% concordance in the paired UC and whole blood samples. Minor allele frequencies of the two SNPs indicated that no maternal gDNA contamination occurred in the genotyping of the UC samples.
Conclusions
gDNAs from all three sources are suitable for standard PCR and pyrosequencing assays. Given that UC provide good quality and quantity gDNA with 100% concordance in the genetic analysis with whole blood, it can replace blood sampling from premature infants. This is likely to reduce the stress and potential side effects associated with invasive sample collection and thus, greatly facilitate participant recruitment for genetic studies.
doi:10.1186/1471-2156-14-105
PMCID: PMC3817355  PMID: 24168095
Premature newborns; Single nucleotide polymorphism; Newborn dried blood spots; Umbilical cord; Genomic DNA; Pyrosequencing; Sample storage
7.  GVRG 2011–2012 Research Study Conclusion: Evaluation of DNA Whole Genome Amplification Technologies for Genotyping 
The evolution of genomic technologies is occurring rapidly and often requires large amounts of source DNA. There is an expanded desire to analyze smaller numbers of cells for higher resolution studies as well as to take advantage of large numbers of archived samples (eg. FFPE, serum, etc.). To provide enough material for the latest genomic technologies, whole genome amplification (WGA) has reemerged as an important and necessary technique. The GVRG has evaluated several new WGA products, assessing the quantity and quality of generated samples as well as sample performance on particular current genomic assay platforms.
The GVRG has completed a benchmarking study evaluation of several commercially available WGA kits using different genotyping assays. The WGA kits were tested on six different samples following the manufacturer's protocols. The samples included Coriell DNA from a trio of CEPH individuals, 2 FFPE DNAs, both from the same individual but one newly extracted and the other extracted two years ago, and one sample from the Coriell trio that was fragmented prior to amplification. The WGA kits were from Sigma, NuGen, Qiagen and GE Life Sciences. The six samples were amplified as blind duplicates at several GVRG member lab sites resulting in a total of 78 amplified products.
Quality metrics were performed on the 78 amplified products using several different techniques in order to provide measurements on yield, fragment size, and concentration. Several widely used genotyping methods were used to evaluate the amplification products, including Illumina Human Omni1 Quad Beadchip, TaqMan copy number and SNP genotyping assays, and STR genotyping. Data will be presented on genotyping concordance and loss of heterozygosity checks across the different platforms where possible. Additional data is being presented here as the study conclusion on new FFPE processing kits utilized for Illumina Infinium Beadchips.
PMCID: PMC3630690
8.  GVRG 2011-2012 Research Study Conclusion: Evaluation of DNA Whole Genome Amplification Technologies for Genotyping 
The evolution of genomic technologies is occurring rapidly and often requires large amounts of source DNA. There is also an expanded desire to analyze smaller numbers of cells for higher resolution studies as well as to take advantage of large numbers of archived samples (eg. FFPE, serum, etc.). To provide enough material for the newest genomic technologies, whole genome amplification (WGA) has reemerged as an important and necessary technique. With some new WGA products on the market, we have evaluated the quantity and quality of WGA products generated as well their performance on some of the current genomic applications. The GVRG has completed a benchmarking study evaluation of 6 commercially available WGA kits using several genotyping assays. Utilizing 6 samples, the different WGA kits were tested following the manufacturer's protocols. The samples included Coriell DNA from a trio of CEPH individuals, 2 FFPE DNAs, both from the same individual (1 newly extracted and the other extracted 2 years ago), and 1 sample from the Coriell trio that was fragmented prior to amplification for a total of 6 samples tested. The WGA kits included those from Sigma, NuGen, Qiagen and GE Life Sciences. The 6 samples were amplified as blind duplicates at several GVRG member lab sites resulting in a total of 78 amplified products. Quality metrics were performed on the 78 amplified products using several different techniques in order to provide measurements on yield, fragment size, and concentration. Several widely used genotyping methods were used to evaluate the amplification products, including Illumina Human Omni1 Quad Beadchip, TaqMan copy number and SNP genotyping assays, and STR genotyping. Data will be presented on genotyping concordance and loss of heterozygosity checks across the different platforms where possible. Additional data is being presented here as the study conclusion on new FFPE processing kits utilized for Illumina Infinium Beadchips.
PMCID: PMC3630547
9.  Comparison of buccal and blood-derived canine DNA, either native or whole genome amplified, for array-based genome-wide association studies 
BMC Research Notes  2011;4:226.
Background
The availability of array-based genotyping platforms for single nucleotide polymorphisms (SNPs) for the canine genome has expanded the opportunities to undertake genome-wide association (GWA) studies to identify the genetic basis for Mendelian and complex traits. Whole blood as the source of high quality DNA is undisputed but often proves impractical for collection of the large numbers of samples necessary to discover the loci underlying complex traits. Further, many countries prohibit the collection of blood from dogs unless medically necessary thereby restricting access to critical control samples from healthy dogs. Alternate sources of DNA, typically from buccal cytobrush extractions, while convenient, have been suggested to have low yield and perform poorly in GWA. Yet buccal cytobrushes provide a cost-effective means of collecting DNA, are readily accepted by dog owners, and represent a large resource base in many canine genetics laboratories. To increase the DNA quantities, whole genome amplification (WGA) can be performed. Thus, the present study assessed the utility of buccal-derived DNA as well as whole genome amplification in comparison to blood samples for use on the most recent iteration of the canine HD SNP array (Illumina).
Findings
In both buccal and blood samples, whether whole genome amplified or not, 97% of the samples had SNP call rates in excess of 80% indicating that the vast majority of the SNPs would be suitable to perform association studies regardless of the DNA source. Similarly, there were no significant differences in marker intensity measurements between buccal and blood samples for copy number variations (CNV) analysis.
Conclusions
All DNA samples assayed, buccal or blood, native or whole genome amplified, are appropriate for use in array-based genome-wide association studies. The concordance between subsets of dogs for which both buccal and blood samples, or those samples whole genome amplified, was shown to average >99%. Thus, the two DNA sources were comparable in the generation of SNP genotypes and intensity values to estimate structural variation indicating the utility for the use of buccal cytobrush samples and the reliability of whole genome amplification for genome-wide association and CNV studies.
doi:10.1186/1756-0500-4-226
PMCID: PMC3145587  PMID: 21718521
10.  Assessing the utility of whole-genome amplified serum DNA for array-based high throughput genotyping 
BMC Genetics  2009;10:85.
Background
Whole genome amplification (WGA) offers new possibilities for genome-wide association studies where limited DNA samples have been collected. This study provides a realistic and high-precision assessment of WGA DNA genotyping performance from 20-year old archived serum samples using the Affymetrix Genome-Wide Human SNP Array 6.0 (SNP6.0) platform.
Results
Whole-genome amplified (WGA) DNA samples from 45 archived serum replicates and 5 fresh sera paired with non-amplified genomic DNA were genotyped in duplicate. All genotyped samples passed the imposed QC thresholds for quantity and quality. In general, WGA serum DNA samples produced low call rates (45.00 +/- 2.69%), although reproducibility for successfully called markers was favorable (concordance = 95.61 +/- 4.39%). Heterozygote dropouts explained the majority (>85% in technical replicates, 50% in paired genomic/serum samples) of discordant results. Genotyping performance on WGA serum DNA samples was improved by implementation of Corrected Robust Linear Model with Maximum Likelihood Classification (CRLMM) algorithm but at the loss of many samples which failed to pass its quality threshold. Poor genotype clustering was evident in the samples that failed the CRLMM confidence threshold.
Conclusions
We conclude that while it is possible to extract genomic DNA and subsequently perform whole-genome amplification from archived serum samples, WGA serum DNA did not perform well and appeared unsuitable for high-resolution genotyping on these arrays.
doi:10.1186/1471-2156-10-85
PMCID: PMC2803178  PMID: 20021669
11.  Whole-genome amplified DNA from stored dried blood spots is reliable in high resolution melting curve and sequencing analysis 
BMC Medical Genetics  2011;12:22.
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.
doi:10.1186/1471-2350-12-22
PMCID: PMC3045282  PMID: 21306642
12.  Phenylketonuria screening registry as a resource for population genetic studies 
Journal of Medical Genetics  2005;42(10):e60.
Background: Neonatal screening for metabolic diseases, involving samples stored on filter paper (Guthrie spots), provides a potential resource for genetic epidemiological studies.
Objective: To develop a method to make these dried blood spots available for large scale genetic epidemiology.
Methods: DNA from untraceable Guthrie spots was extracted using a saponin and chelex-100 based method and preamplified by improved primer preamplification. Analyses were done on 38 samples each of fresh, 10, and 25 year old Guthrie spots and the success rate determined for PCR amplification for five amplicon lengths.
Results: The method was applicable even on 25 year old samples. The success rate was 100% for 100 bp amplicons and 80% for 396 bp amplicons. Ninety four Guthrie samples were genotyped, including carriers of two different PKU mutations; all carriers were found (six R158Q, four R252W), with no false positives. Finally, 2132 anonymous samples from the Swedish PKU registry were extracted and preamplified and the allele frequencies of APOε4, PPARγ Pro12Ala, and the CCR5 32 bp deletion determined. Local variations in allele frequencies suggested subpopulation structuring. There was a significant difference (p<0.01) in regional allele frequencies for the CCR5 32 bp deletion in the Swedish population.
Conclusion: Whole genome amplification makes it feasible to conduct large genetic epidemiological studies using PKU screening registries.
doi:10.1136/jmg.2005.032987
PMCID: PMC1735924  PMID: 16199543
13.  Evaluation of DNA Whole Genome Amplification Technologies for Genotyping 
The evolution of genomic technologies is occurring rapidly and often requires large amounts of source DNA. There is also an expanded desire to analyze smaller numbers of cells for higher resolution studies as well as to take advantage of large numbers of archived samples (e.g. FFPE, serum, etc.). To provide enough material for the newest genomic technologies, whole genome amplification (WGA) has reemerged as an important and necessary technique. With some new WGA products on the market, we have evaluated the quantity and quality of WGA products generated as well their performance on some of the current genomic applications. The GVRG has completed a benchmarking study evaluation of 6 commercially available WGA kits using several genotyping assays. Utilizing 6 samples, the different WGA kits were tested following the manufacturer's protocols. The samples included Coriell DNA from a trio of CEPH individuals, 2 FFPE DNAs, both from the same individual (1 newly extracted and the other extracted 2 years ago), and 1 sample from the Coriell trio that was fragmented prior to amplification for a total of 6 samples tested. The WGA kits included those from Sigma, NuGen, Qiagen and GE Life Sciences. The 6 samples were amplified as blind duplicates at several GVRG member lab sites resulting in a total of 78 amplified products. Quality metrics were performed on the 78 amplified products using several different techniques in order to provide measurements on yield, fragment size, and concentration. Several widely used genotyping methods were used to evaluate the amplification products, including Illumina Human Omni1 Quad Beadchip, TaqMan copy number and SNP genotyping assays, and STR genotyping. Data will be presented on genotyping concordance and loss of heterozygosity checks across the different platforms where possible.
PMCID: PMC3186474
14.  Human Cytomegalovirus: detection of congenital and perinatal infection in Argentina 
BMC Pediatrics  2004;4:11.
Background
Human cytomegalovirus (CMV) is one of the most commonly found agents of congenital infections. Primary maternal infection is associated with risk of symptomatic congenital diseases, and high morbidity is frequently associated with very low birth weight. Neonates with asymptomatic infection develop various sequelae during infancy. This is the first Argentine study performed in neonates with congenital and postnatal HCMV infection. The purpose of this study was to evaluate the performance of the polymerase chain reaction (PCR) technique with different pairs of primers, to detect cytomegalovirus isolated in tissue cultures and directly in urine and dried blood spot (DBS) specimens. Results were compared with IgM detection.
Methods
The study was performed between 1999 and 2001 on routine samples in the Laboratory. A total of 61 urine and 56 serum samples were selected from 61 newborns/infants, 33 patients whose samples were analyzed during the first two to three weeks of life were considered congenital infections; the remaining 28 patients whose samples were taken later than the third week were grouped as perinatal infections, although only in 4 the perinatal transmission of infection was determined unequivocally
Cytomegalovirus diagnosis was made by isolating the virus from urine samples in human foreskin fibroblast cells.
Three different primer pairs directed to IE, LA and gB genes were used for the HCMV PCR assay in viral isolates. Subsequently, PCR and nested PCR (nPCR) assays with gB primers were performed directly in urine and in 11 samples of dried blood spot (DBS) on Guthrie Card, these results were then compared with serology.
Results
The main clinical manifestations of the 33 patients with congenital infection were purpura, jaundice, hepatomegaly and anaemia. Three patients presented low birth weight as single symptom, 10, intracranial calcifications, and 2, kidney failure. In the 28 patients grouped as with perinatal infection, anaemia, hepatosplenomegaly and enzymatic alteration were predominant, and 4 patients were HIV positive.
The primers used to amplify the gB region had a PCR positivity rate of 100%, whereas those that amplified IE and LA regions had a PCR positivity rate of 54% and 61% respectively, in CMV isolates.
Amplification by PCR of urine samples (with no previous DNA extraction), using primers for the gB region, detected 34/61 positive samples. Out of the 33 samples from patients with congenital infection, 24 (73%) were positive. When nPCR was used in these samples, all were positive, whereas in the remaining 28 patients, two negative cases were found.
Cytomegalovirus DNA detection in 11 samples was also carried out in DBS: 7 DBS samples were positive and 4 were negative.
Conclusions
Primers directed to the gB fragment region were the best choice for the detection of CMV DNA in positive isolates. In congenital infections, direct PCR in urine was positive in a high percentage (73%) of samples; however, in patients grouped as with perinatal infection only 36% of the cases were positive. With n-PCR, total sample positivity reached 97%. PCR technique performed in DBS allowed identifying congenital infection in four patients and to be confirmed in 3. These results show the value of nPCR for the detection of all cases of CMV infection. The assay offers the advantage that it may be performed within the normal working day and provides reliable results in a much shorter time frame than that required for either traditional tissue culture or the shell-viral assay.
doi:10.1186/1471-2431-4-11
PMCID: PMC449715  PMID: 15214967
15.  Superparamagnetic-bead Based Method: An Effective DNA Extraction from Dried Blood Spots (DBS) for Diagnostic PCR 
Introduction: Storing blood as dried spots on filter paper is a trustworthy approach used in genetic screening issues which justifies the necessity for a reliable DNA extraction method. The present work aims to investigate the effectiveness of superparamagnetic-bead based method in extracting DNA from dried blood spots (DBS).
Materials and Methods: Sixteen venous blood samples collected in K3-EDTA tubes (400μl of whole blood) were used for the spotting (4 circles each 100μl) on Ahlstrom 226 grad filter papers, for extraction and comparison. To ensure effectiveness, the extracted DNA was checked for quantity using the Quant-iT™ dsDNA Broad-Range Assay Kit and for quality by polymerase chain reaction (PCR) amplification of 344 bp segment of the HBB gene. Hybridization assays based on the dynamic allele specific hybridization (DASH) technique for two hemoglobin beta (HBB) mutations in genomic DNA extracted from DBS of ß-thalassemia patients were also performed to ensure the quality of extraction.
Results: The results revealed a compatible effectiveness of the superparamagnetic-bead based method in extracting DNA from DBS particularly when incubating the DBS with lysis buffers BL+BLM overnight. A mean concentration of 21ng/ μl was obtained with lysis buffers BL+BLM overnight incubation compared to 5.2 ng/μl for 2 h incubation with lysis buffers BL+BLM and 4.7 ng/μl when extraction performed using the lysis buffer BLM alone. Moreover, PCR amplification of 344 bp segment of the HBB showed a good quality of the extracted DNA.
Conclusion: It was concluded that the superparamagnetic-bead based method is a reliable and effective method for DNA extraction from DBS and can be adopted for genetic diagnostic purposes.
doi:10.7860/JCDR/2014/8171.4226
PMCID: PMC4064838  PMID: 24959449
Superparamagnetic-beads; DNA extraction; Dried blood spots; PCR
16.  A microarray platform and novel SNP calling algorithm to evaluate Plasmodium falciparum field samples of low DNA quantity 
BMC Genomics  2014;15(1):719.
Background
Analysis of single nucleotide polymorphisms (SNPs) derived from whole-genome studies allows for rapid evaluation of genome-wide diversity, and genomic epidemiology studies of Plasmodium falciparum provide insights into parasite population structure, gene flow, drug resistance and vaccine development. In areas with adequate cold chain facilities, large volumes of leukocyte-depleted patient blood can be frozen for use in parasite genomic analyses. In more remote endemic areas smaller volumes of infected blood are taken by finger prick, and dried and stored on filter paper. These dried blood spots do not generally yield enough concentrated parasite DNA for whole-genome sequencing.
Results
A DNA microarray was designed for use on field samples to type a genome-wide set of SNPs which prior sequencing had shown to be variable in Africa, Southeast Asia, and Papua New Guinea. An algorithm was designed to call SNPs in samples with low parasite DNA. With this new algorithm SNP-calling accuracy of 98% was measured by hybridizing purified DNA from malaria lab strains and comparing calls with SNPs called from full genome sequences. An average accuracy of >98% was likewise obtained for DNA extracted from malaria field samples collected in studies in Southeast Asia, with an average call rate of > 82%.
Conclusion
This new high-density microarray provided high quality SNP calls from a wide range of parasite DNA quantities, and represents a robust tool for genome-wide analysis of malaria parasites in diverse settings.
doi:10.1186/1471-2164-15-719
PMCID: PMC4153902  PMID: 25159520
Plasmodium falciparum; Malaria; Microarray
17.  Optimized DNA extraction from neonatal dried blood spots: application in methylome profiling 
BMC Biotechnology  2014;14:60.
Background
Neonatal dried blood spots (DBS) represent an inexpensive method for long-term biobanking worldwide and are considered gold mines for research for several human diseases, including those of metabolic, infectious, genetic and epigenetic origin. However, the utility of DBS is restricted by the limited amount and quality of extractable biomolecules (including DNA), especially for genome wide profiling. Degradation of DNA in DBS often occurs during storage and extraction. Moreover, amplifying small quantities of DNA often leads to a bias in subsequent data, particularly in methylome profiles. Thus it is important to develop methodologies that maximize both the yield and quality of DNA from DBS for downstream analyses.
Results
Using combinations of in-house-derived and modified commercial extraction kits, we developed a robust and efficient protocol, compatible with methylome studies, many of which require stringent bisulfite conversion steps. Several parameters were tested in a step-wise manner, including blood extraction, cell lysis, protein digestion, and DNA precipitation, purification and elution. DNA quality was assessed based on spectrophotometric measurements, DNA detectability by PCR, and DNA integrity by gel electrophoresis and bioanalyzer analyses. Genome scale Infinium HumanMethylation450 and locus-specific pyrosequencing data generated using the refined DBS extraction protocol were of high quality, reproducible and consistent.
Conclusions
This study may prove useful to meet the increased demand for research on prenatal, particularly epigenetic, origins of human diseases and for newborn screening programs, all of which are often based on DNA extracted from DBS.
doi:10.1186/1472-6750-14-60
PMCID: PMC4086704  PMID: 24980254
Blood spot; DNA extraction; Epigenetics; Methylome; HM450; Pyrosequencing; Whole bisulfitome amplification; QIAamp; GenSolve; NucleoSpin
18.  Apo E genotyping from blood stored on filter paper 
Background & objectives:
Dried blood spotted on to filter paper has been found suitable for a large number of studies. In tropical countries with varying temperature conditions the use of dried blood needs to be validated. We carried out this study to assess the use of blood spotted filter paper as a transport system to study genotyping of Apo E gene.
Methods:
Fifty five patients visiting Cardiothoracic Neuroscience Centre (CNC) OPD at the All India Institute of Medical Sciences (AIIMS), New Delhi, and referred for lipid investigations to Cardiac Biochemistry Laboratory were selected at random. Blood was spotted on to Whatman 3 MM filter paper, dried and stored at room temperature. Genomic DNA was extracted and genotyping was carried out at the end of 0, 3 and 12 months. The study was further validated using samples collected on to filter paper from four centres and stored for eight years at room temperature. The temperature and humidity conditions of the centre varied widely.
Results:
Fifty five samples collected on to filter paper showed exact match of the genotyping when compared to fresh blood. In dried blood samples collected and stored for 1 yr at room temperature DNA extraction and apo E genotyping was done successfully.
Interpretation & conclusions:
The present results showed the feasibility of using dried blood samples on filter paper for apo E genotyping in tropical temperature. The findings need to be validated on a large sample before being recommended for use.
PMCID: PMC3361867  PMID: 22561617
19.  Feasibility of Detecting Human Immunodeficiency Virus Type 1 Drug Resistance in DNA Extracted from Whole Blood or Dried Blood Spots▿  
Journal of Clinical Microbiology  2007;45(10):3342-3351.
Due to high cost, availability of human immunodeficiency virus type 1 (HIV-1) drug resistance testing in resource-poor settings is still limited. We therefore evaluated the usefulness of viral DNA extracted from either whole blood or dried blood spots (DBS). Samples were collected from 50 patients receiving therapy and 10 therapy-naïve patients. Amplification and sequencing of RNA and DNA was performed using an in-house assay. Protease (PR) and reverse transcriptase (RT) sequences of plasma viral RNA were obtained for 96.6% and 89.7%, respectively, of the 29 patients with a detectable viral load. For cellular viral DNA, useful PR and RT sequences were obtained for 96.6% and 93.1% of the whole-blood-cell samples and for 93.1% and 93.1% of the DBS samples, respectively. For the 31 patients with an undetectable viral load, PR and RT sequences were obtained for 67.7% and 61.3% of the whole-blood-cell DNA preparations and for 54.8% and 58.1% of the DBS DNA preparations, respectively. A good correlation between RNA and DNA sequences was found; most discordances were caused by the detection of mixed amino acids. Of the RT drug-resistant mutations, 13 (38.2%) were seen in RNA only, 6 (17.6%) in DNA only, and 15 (44.1%) in both. Repeated amplification and sequencing of DNA extracts revealed a lack of reproducibility for the detection of drug resistance mutations in a number of samples, indicating a possible founder effect. In conclusion, this study shows the feasibility of genotypic drug resistance testing on whole blood cells or DBS and its possible usefulness for HIV-1 subtyping or examining the overall distribution of drug resistance in a population. For individual patients, RNA sequencing was shown to be superior to DNA sequencing, especially for patients who experienced early treatment failure. The use of DNA extracted from whole blood or DBS for the detection of archived drug resistance mutations deserves further study.
doi:10.1128/JCM.00814-07
PMCID: PMC2045371  PMID: 17670924
20.  Detection of Duck Hepatitis B Virus DNA on Filter Paper by PCR and SYBR Green Dye-Based Quantitative PCR 
Journal of Clinical Microbiology  2002;40(7):2584-2590.
Duck hepatitis B virus (DHBV) belongs to the Hepadnaviridae family, which includes human Hepatitis B virus (HBV) and Woodchuck hepatitis virus. It is widely distributed in wild and domestic ducks due to congenital transmission. HBV is a worldwide health problem, with carriers at risk of developing cirrhosis and liver cancer. Medical staff and scientists working with HBV must be vaccinated because of its contagious nature. DHBV is a safe surrogate for HBV because of their similarities. Collection of serum and blood samples on filter paper has been used to screen for metabolic disorders, genetic diseases, and viral infection and for evolutionary studies of the genome. In this study, DHBV from serum and blood dried on filters was detected by PCR. A 0.1-μl sample was sufficient for detection. The immobilization potential of filter papers for DHBV was examined, and the highest yield of PCR products was observed with Whatman paper. Dried serum was stable under different storage temperatures for 4 weeks, but the yields of PCR products decreased when the temperature was ⩾4°C. The optimal condition for storage was −70°C. A newly developed quantitative PCR based on monitoring the amplification by measuring the increase in fluorescence caused by the binding of SYBR green dye to double-stranded products was applied herein. DHBV genomic DNA cloned in a plasmid was used for the generation of standard DHBV DNA for quantitative PCR. It validated results from PCR in terms of the copy number of DHBV particles. The specificity of PCR was demonstrated by melting curve analysis, and the differentiation of two DHBV isolates amplified from dried serum was demonstrated based on their melting temperatures determined by GC contents and sequence. It was easier and simpler than other PCR-based DNA techniques. The use of serum dried on filters allows samples from distant field for which cold storage and transportation are a problem to be mailed to the diagnostic laboratory. Samples can be archived for comparison and used as a source of DNA for cloning and sequencing.
doi:10.1128/JCM.40.7.2584-2590.2002
PMCID: PMC120600  PMID: 12089280
21.  Evaluation of Different Cytomegalovirus (CMV) DNA PCR Protocols for Analysis of Dried Blood Spots from Consecutive Cases of Neonates with Congenital CMV Infections▿  
Journal of Clinical Microbiology  2008;46(3):943-946.
Two protocols for the extraction of cytomegalovirus (CMV) DNA and two methods for the amplification of CMV DNA in dried blood spots were evaluated for the retrospective diagnosis of congenital CMV infection. During the period from 1996 to 2006, a urine screening program detected 76 congenitally infected neonates. Stored Guthrie cards with blood from 55 cases and 12 controls were tested. Two spots of dried blood were cut from each card and evaluated in two centers. CMV DNA was extracted from a whole single spot. Center 1 used phenol-chloroform extraction and ethanol precipitation followed by a conventional PCR. Center 2 used the NucliSens easyMAG automated DNA/RNA extraction platform (bioMérieux) followed by a real-time PCR. For evaluation of the extraction method, DNA extracted from each blood spot was evaluated by the amplification method used by the collaborating center. The sensitivities were 66% for center 1 and 73% for center 2. None of the controls were positive. A sensitivity as high as 82% could be obtained by combining the most sensitive extraction method (the phenol-chloroform procedure) with the most sensitive PCR method (real-time PCR). The detection rate was not influenced by the duration of storage of the spots. The sensitivity was higher with blood from congenitally infected cases due to a primary maternal CMV infection, regardless of the protocol used. However, the difference reached significance only for the least-sensitive protocol (P = 0.036).
doi:10.1128/JCM.01391-07
PMCID: PMC2268368  PMID: 18199787
22.  Single-tube-genotyping of gastric cancer related SNPs by directly using whole blood and paper-dried blood as starting materials 
AIM: To demonstrate an inexpensive method for typing gastric cancer related single nucleotide polymorphisms (SNPs) using whole blood or paper-dried blood as starting materials.
METHODS: PCR amplification is directly carried out from the whole blood or paper-dried blood sample without any DNA extraction step. Before PCR, a blood sample, four primers, and all of biological reagents necessary for PCR were added at a time; After PCR, the amplified products were directly separated by slab gel electrophoresis or microchip CE without any purification. SNP typing was performed by tetra-primer PCR with two inner primers specific to each allele and two outer primers defining the length of allele-specific amplicons. Genotypes were directly discriminated by the size of amplicons specific to each allele, thereby avoiding any post-PCR process.
RESULTS: Using a special PCR buffer, inhibitory substances in blood (including the anticoagulant in blood) and filter paper were effectively suppressed; a “true” single-tube-genotyping is thus realized. We successfully determined genotypes IL-1B-511 and IL-1B-31 polymorphisms at the gene IL-1B by using whole-blood and paper-dried blood samples as starting materials respectively. The method is so sensitive that 0.5-1.0 μL of blood sample is enough to give a satisfactory typing results. The genotyping results were confirmed by RFLP-PCR using purified genome DNA, indicating that amplification specificity was not affected by inhibitory components (including coagulants) in blood or filter paper.
CONCLUSION: Compared with SNP typing methods based on purified DNA, the proposed method is labor-saving, simple, inexpensive, and less cross-contaminated. It is promising to use this method to type other SNPs.
doi:10.3748/wjg.v12.i24.3814
PMCID: PMC4087927  PMID: 16804964
Gastric cancer; Whole blood; Paper-dried blood; Tetra-PCR; SNP; IL-1B-31; IL-1B-511
23.  Stability of dried blood spot specimens for detection of human immunodeficiency virus DNA by polymerase chain reaction. 
Journal of Clinical Microbiology  1992;30(12):3039-3042.
Blood sampling on filter paper has many advantages for the detection of perinatal human immunodeficiency virus (HIV) infection by the polymerase chain reaction (PCR). However, if the method is to be widely used, an assessment of its performance under field conditions is required. To simulate conditions in the field, 50-microliters aliquots of whole blood containing low levels of HIV proviral DNA (4 to 1,024 copies per 100,000 nucleated cells) were spotted onto filter paper; dried; and subjected to heat, humidity, and prolonged storage at room temperature. After exposure, the DNA was recovered and amplified with primers to human leukocyte antigen DQ alpha- and HIV-specific sequences. Treatment at 37 degrees C and 60% humidity for 7 days, storage for 12 weeks at 22 degrees C, and freeze-thawing twice had no adverse effect on PCR reactivity when compared with the results obtained with reference spots stored at -20 degrees C. The lower limits of HIV detection in all tests ranged from 4 to 16 HIV copies per 100,000 cells. Fixation in 70% ethanol improved the amplification of low levels of HIV DNA and reduced biohazard risks. These findings suggest that dried blood spots will provide a powerful new resource for testing for HIV by PCR, especially in remote areas where refrigeration and immediate sample processing are unavailable.
PMCID: PMC270585  PMID: 1452682
24.  The design and evaluation of a shaped filter collection device to sample and store defined volume dried blood spots from finger pricks 
Malaria Journal  2015;14:45.
Background
Dried blood spots are a common medium for collecting patient blood prior to testing for malaria by molecular methods. A new shaped filter device for the quick and simple collection of a designated volume of patient blood has been designed and tested against conventional blood spots for accuracy and precision.
Methods
Shaped filter devices were laser cut from Whatman GB003 paper to absorb a 20 μl blood volume. These devices were used to sample Plasmodium falciparum infected blood and the volume absorbed was measured volumetrically. Conventional blood spots were made by pipetting 20 μl of the same blood onto Whatman 3MM paper. DNA was extracted from both types of dried blood spot using Qiagen DNA blood mini or Chelex extraction for real-time PCR analysis, and PURE extraction for malaria LAMP testing.
Results
The shaped filter devices collected a mean volume of 21.1 μl of blood, with a coefficient of variance of 8.1%. When used for DNA extraction by Chelex and Qiagen methodologies the mean number of international standard units of P. falciparum DNA recovered per μl of the eluate was 53.1 (95% CI: 49.4 to 56.7) and 32.7 (95% CI: 28.8 to 36.6), respectively for the shaped filter device, and 54.6 (95% CI: 52.1 to 57.1) and 12.0 (95% CI: 9.9 to 14.1), respectively for the 3MM blood spots. Qiagen extraction of 200 μl of whole infected blood yielded 853.6 international standard units of P. falciparum DNA per μl of eluate.
Conclusions
A shaped filter device provides a simple way to quickly sample and store a defined volume of blood without the need for any additional measuring devices. Resultant dried blood spots may be employed for DNA extraction using a variety of technologies for nucleic acid amplification without the need for repeated cleaning of scissors or punches to prevent cross contamination of samples and results are comparable to traditional DBS.
Electronic supplementary material
The online version of this article (doi:10.1186/s12936-015-0558-x) contains supplementary material, which is available to authorized users.
doi:10.1186/s12936-015-0558-x
PMCID: PMC4327959  PMID: 25652763
Dried blood spot; Malaria; Sampling; Shaped filter paper; DNA extraction; PCR; LAMP
25.  Real-time PCR detection of Plasmodium directly from whole blood and filter paper samples 
Malaria Journal  2011;10:244.
Background
Real-time PCR is a sensitive and specific method for the analysis of Plasmodium DNA. However, prior purification of genomic DNA from blood is necessary since PCR inhibitors and quenching of fluorophores from blood prevent efficient amplification and detection of PCR products.
Methods
Reagents designed to specifically overcome PCR inhibition and quenching of fluorescence were evaluated for real-time PCR amplification of Plasmodium DNA directly from blood. Whole blood from clinical samples and dried blood spots collected in the field in Colombia were tested.
Results
Amplification and fluorescence detection by real-time PCR were optimal with 40× SYBR® Green dye and 5% blood volume in the PCR reaction. Plasmodium DNA was detected directly from both whole blood and dried blood spots from clinical samples. The sensitivity and specificity ranged from 93-100% compared with PCR performed on purified Plasmodium DNA.
Conclusions
The methodology described facilitates high-throughput testing of blood samples collected in the field by fluorescence-based real-time PCR. This method can be applied to a broad range of clinical studies with the advantages of immediate sample testing, lower experimental costs and time-savings.
doi:10.1186/1475-2875-10-244
PMCID: PMC3171379  PMID: 21851640

Results 1-25 (1224861)