Document Detail


A microfluidic liquid phase nucleic acid purification chip to selectively isolate DNA or RNA from low copy/single bacterial cells in minute sample volume followed by direct on-chip quantitative PCR assay.
MedLine Citation:
PMID:  23272769     Owner:  NLM     Status:  Publisher    
Abstract/OtherAbstract:
Purification of nucleic acids from low quantity of bacterial cells in minute volume is im- portant in many clinical and biological applications. We developed a novel microfluidic liquid phase nucleic acid purification chip to selectively isolate DNA or RNA from bacterial cell in the range of 5000 down to a single cell in the sample volume of 1 µl or 125 nl, which can be directly put through on-chip quantitative PCR assay. The aqueous phase bacterial lysate was isolated in an array of micro-wells, after which an immiscible organic (phenol-chloroform) phase was introduced in a headspace channel connecting the micro-well array. Continuous flow of the organic phase increases the interfacial contact with the aqueous phase to achieve purification of target nucleic acid through phase partitioning. Significantly enhanced nucleic acid recovery yield up to 10 folds higher was achieved using the chip based liquid phase nucleic acid purification technique compared to that obtained by conventional column based solid phase nucleic acid extraction method. One step vacuum driven microfluidics allowed on-chip quantitative PCR assay to be carried out in the same micro-wells within which bacterial nucleic acids were isolated, avoiding sample loss during liquid transfer. Using this nucleic acid purifi- cation device set in a two-dimensional array format of 900 micro-wells, it was demonstrated for the first time that high throughput extraction of RNA couple with direct on-chip PCR analysis from single bacterial cells could be achieved. Our microfluidic platform offered a simple and effective solution for nucleic acid preparation, which can be integrated for automated bacterial pathogen detection and high throughput transcriptional profiling.
Authors:
Rui Zhang; Haiqing Gong; Xudong Zeng; Chaoping Lou; Chun Chau Sze
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Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2012-12-31
Journal Detail:
Title:  Analytical chemistry     Volume:  -     ISSN:  1520-6882     ISO Abbreviation:  Anal. Chem.     Publication Date:  2012 Dec 
Date Detail:
Created Date:  2012-12-31     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  0370536     Medline TA:  Anal Chem     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
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