Optimization of a simple, automatable extraction method to recover sufficient DNA from low copy number DNA samples for generation of short tandem repeat profiles

Croat Med J. 2005 Aug;46(4):578-86.

Abstract

Aim: To develop an automated, high throughput extraction protocol in order to produce database eligible profiles from fingerprints and other low copy number (LCN) DNA sources.

Methods: Extraction of either purified control DNA or buccal cells, for example, with commercial kits was compared to extraction with a simple digestion buffer and a subsequent concentration and purification. Results were evaluated based on the amount of DNA recovered and the completeness of the DNA profiles produced.

Results: Simple procedures with fewer steps were superior to commercial kits, such as DNA IQ (Promega, Madison, WI, USA) and QiaAmp (Qiagen, Valencia, CA, USA), and other protocols with many manipulations. The optimized protocol included a thirty-minute incubation with 0.01% SDS and proteinase K at 56 degrees C, followed by an incubation at 100 degrees C for 10 minutes. Concentration of the extract and removal of the SDS was accomplished with a Microcon 100 (Millipore, Bedford, MA, USA), which can be assembled into a 96 well plate, the Microcon-96 Retentate Assembly Plate (Millipore) for automation. The addition of 1 ng Poly A RNA to the Microcon significantly improved DNA recovery.

Conclusion: A one-step sample digestion followed by sample concentration/purification minimized sample loss and maximized amplification input. Moreover, this methodology can be easily adapted for automation. Implementation of this protocol, due to the numerous potential sources of LCN DNA samples, will enhance the recovery of biological evidence from crime scenes and may be a source of database profiles.

MeSH terms

  • Automation
  • DNA / genetics
  • DNA / isolation & purification*
  • DNA / supply & distribution
  • DNA Fingerprinting / methods*
  • Forensic Medicine
  • Humans
  • Tandem Repeat Sequences / genetics*
  • United States

Substances

  • DNA