Cargando…

Effects of DNA mass on multiple displacement whole genome amplification and genotyping performance

BACKGROUND: Whole genome amplification (WGA) promises to eliminate practical molecular genetic analysis limitations associated with genomic DNA (gDNA) quantity. We evaluated the performance of multiple displacement amplification (MDA) WGA using gDNA extracted from lymphoblastoid cell lines (N = 27)...

Descripción completa

Detalles Bibliográficos
Autores principales: Bergen, Andrew W, Qi, Ying, Haque, Kashif A, Welch, Robert A, Chanock, Stephen J
Formato: Texto
Lenguaje:English
Publicado: BioMed Central 2005
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1249558/
https://www.ncbi.nlm.nih.gov/pubmed/16168060
http://dx.doi.org/10.1186/1472-6750-5-24
_version_ 1782125712818307072
author Bergen, Andrew W
Qi, Ying
Haque, Kashif A
Welch, Robert A
Chanock, Stephen J
author_facet Bergen, Andrew W
Qi, Ying
Haque, Kashif A
Welch, Robert A
Chanock, Stephen J
author_sort Bergen, Andrew W
collection PubMed
description BACKGROUND: Whole genome amplification (WGA) promises to eliminate practical molecular genetic analysis limitations associated with genomic DNA (gDNA) quantity. We evaluated the performance of multiple displacement amplification (MDA) WGA using gDNA extracted from lymphoblastoid cell lines (N = 27) with a range of starting gDNA input of 1–200 ng into the WGA reaction. Yield and composition analysis of whole genome amplified DNA (wgaDNA) was performed using three DNA quantification methods (OD, PicoGreen(® )and RT-PCR). Two panels of N = 15 STR (using the AmpFlSTR(® )Identifiler(® )panel) and N = 49 SNP (TaqMan(®)) genotyping assays were performed on each gDNA and wgaDNA sample in duplicate. gDNA and wgaDNA masses of 1, 4 and 20 ng were used in the SNP assays to evaluate the effects of DNA mass on SNP genotyping assay performance. A total of N = 6,880 STR and N = 56,448 SNP genotype attempts provided adequate power to detect differences in STR and SNP genotyping performance between gDNA and wgaDNA, and among wgaDNA produced from a range of gDNA templates inputs. RESULTS: The proportion of double-stranded wgaDNA and human-specific PCR amplifiable wgaDNA increased with increased gDNA input into the WGA reaction. Increased amounts of gDNA input into the WGA reaction improved wgaDNA genotyping performance. Genotype completion or genotype concordance rates of wgaDNA produced from all gDNA input levels were observed to be reduced compared to gDNA, although the reduction was not always statistically significant. Reduced wgaDNA genotyping performance was primarily due to the increased variance of allelic amplification, resulting in loss of heterozygosity or increased undetermined genotypes. MDA WGA produces wgaDNA from no template control samples; such samples exhibited substantial false-positive genotyping rates. CONCLUSION: The amount of gDNA input into the MDA WGA reaction is a critical determinant of genotyping performance of wgaDNA. At least 10 ng of lymphoblastoid gDNA input into MDA WGA is required to obtain wgaDNA TaqMan(® )SNP assay genotyping performance equivalent to that of gDNA. Over 100 ng of lymphoblastoid gDNA input into MDA WGA is required to obtain optimal STR genotyping performance using the AmpFlSTR(® )Identifiler(® )panel from wgaDNA equivalent to that of gDNA.
format Text
id pubmed-1249558
institution National Center for Biotechnology Information
language English
publishDate 2005
publisher BioMed Central
record_format MEDLINE/PubMed
spelling pubmed-12495582005-10-08 Effects of DNA mass on multiple displacement whole genome amplification and genotyping performance Bergen, Andrew W Qi, Ying Haque, Kashif A Welch, Robert A Chanock, Stephen J BMC Biotechnol Methodology Article BACKGROUND: Whole genome amplification (WGA) promises to eliminate practical molecular genetic analysis limitations associated with genomic DNA (gDNA) quantity. We evaluated the performance of multiple displacement amplification (MDA) WGA using gDNA extracted from lymphoblastoid cell lines (N = 27) with a range of starting gDNA input of 1–200 ng into the WGA reaction. Yield and composition analysis of whole genome amplified DNA (wgaDNA) was performed using three DNA quantification methods (OD, PicoGreen(® )and RT-PCR). Two panels of N = 15 STR (using the AmpFlSTR(® )Identifiler(® )panel) and N = 49 SNP (TaqMan(®)) genotyping assays were performed on each gDNA and wgaDNA sample in duplicate. gDNA and wgaDNA masses of 1, 4 and 20 ng were used in the SNP assays to evaluate the effects of DNA mass on SNP genotyping assay performance. A total of N = 6,880 STR and N = 56,448 SNP genotype attempts provided adequate power to detect differences in STR and SNP genotyping performance between gDNA and wgaDNA, and among wgaDNA produced from a range of gDNA templates inputs. RESULTS: The proportion of double-stranded wgaDNA and human-specific PCR amplifiable wgaDNA increased with increased gDNA input into the WGA reaction. Increased amounts of gDNA input into the WGA reaction improved wgaDNA genotyping performance. Genotype completion or genotype concordance rates of wgaDNA produced from all gDNA input levels were observed to be reduced compared to gDNA, although the reduction was not always statistically significant. Reduced wgaDNA genotyping performance was primarily due to the increased variance of allelic amplification, resulting in loss of heterozygosity or increased undetermined genotypes. MDA WGA produces wgaDNA from no template control samples; such samples exhibited substantial false-positive genotyping rates. CONCLUSION: The amount of gDNA input into the MDA WGA reaction is a critical determinant of genotyping performance of wgaDNA. At least 10 ng of lymphoblastoid gDNA input into MDA WGA is required to obtain wgaDNA TaqMan(® )SNP assay genotyping performance equivalent to that of gDNA. Over 100 ng of lymphoblastoid gDNA input into MDA WGA is required to obtain optimal STR genotyping performance using the AmpFlSTR(® )Identifiler(® )panel from wgaDNA equivalent to that of gDNA. BioMed Central 2005-09-16 /pmc/articles/PMC1249558/ /pubmed/16168060 http://dx.doi.org/10.1186/1472-6750-5-24 Text en Copyright © 2005 Bergen et al; licensee BioMed Central Ltd.
spellingShingle Methodology Article
Bergen, Andrew W
Qi, Ying
Haque, Kashif A
Welch, Robert A
Chanock, Stephen J
Effects of DNA mass on multiple displacement whole genome amplification and genotyping performance
title Effects of DNA mass on multiple displacement whole genome amplification and genotyping performance
title_full Effects of DNA mass on multiple displacement whole genome amplification and genotyping performance
title_fullStr Effects of DNA mass on multiple displacement whole genome amplification and genotyping performance
title_full_unstemmed Effects of DNA mass on multiple displacement whole genome amplification and genotyping performance
title_short Effects of DNA mass on multiple displacement whole genome amplification and genotyping performance
title_sort effects of dna mass on multiple displacement whole genome amplification and genotyping performance
topic Methodology Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1249558/
https://www.ncbi.nlm.nih.gov/pubmed/16168060
http://dx.doi.org/10.1186/1472-6750-5-24
work_keys_str_mv AT bergenandreww effectsofdnamassonmultipledisplacementwholegenomeamplificationandgenotypingperformance
AT qiying effectsofdnamassonmultipledisplacementwholegenomeamplificationandgenotypingperformance
AT haquekashifa effectsofdnamassonmultipledisplacementwholegenomeamplificationandgenotypingperformance
AT welchroberta effectsofdnamassonmultipledisplacementwholegenomeamplificationandgenotypingperformance
AT chanockstephenj effectsofdnamassonmultipledisplacementwholegenomeamplificationandgenotypingperformance