Rapid multiplex DNA amplification on an inexpensive microdevice for human identification via short tandem repeat analysis.
Anal Chim Acta
; 980: 41-49, 2017 Aug 08.
Article
em En
| MEDLINE
| ID: mdl-28622802
ABSTRACT
Forensic DNA analysis requires several steps, including DNA extraction, PCR amplification, and separation of PCR fragments. Intuitively, there are numerous situations where it would be beneficial to speed up the overall DNA analysis process; in this work, we focus on the most time-consuming component in the analysis pipeline, namely the polymerase chain reaction (PCR). Primers were specially designed to target 10 human genomic loci, all yielding amplicons shorter than 350 bases, for ease of downstream integration with on-board microchip electrophoresis. Primer concentrations were adjusted specifically for microdevice amplification, resulting in well-balanced short tandem repeat (STR) profiles. Furthermore, studies were performed to push the limits of the DNA polymerase to achieve rapid, multiplexed PCR on various substrates, including transparent and black polyethylene terephthalate (Pe), and with two distinct adhesives, toner and heat sensitive adhesive (HSA). Rapid STR-based multiplexed PCR amplification is demonstrated in 15 min on a Pe microdevice using a custom-built system for fluid flow control and thermocycling for the full 10-plex, and in 10 min for a smaller multiplex consisting of six core CODIS loci plus Amelogenin with amplicons shorter than 200bp. Lastly, preliminary studies indicate the capability of this PCR microdevice platform to be integrated with both upstream DNA extraction, and downstream microchip electrophoresis. This, coupled to the use of reagents that are compatible with lyophilization (lyo-compatible) for PCR, represents the potential for a fully integrated rotationally-driven microdevice for complete forensic DNA analysis.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Repetições de Microssatélites
/
Técnicas de Amplificação de Ácido Nucleico
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Eletroforese em Microchip
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Genética Forense
Tipo de estudo:
Diagnostic_studies
Limite:
Humans
Idioma:
En
Revista:
Anal Chim Acta
Ano de publicação:
2017
Tipo de documento:
Article
País de afiliação:
Estados Unidos
País de publicação:
HOLANDA
/
HOLLAND
/
NETHERLANDS
/
NL
/
PAISES BAJOS
/
THE NETHERLANDS