Obtaining High Quality DNA from Diverse Clinical Samples

Rachael Melton‐Kreft1, Tracy Spirk1

1 Center of Excellence in Biofilm Research, Allegheny Singer Research Institute, Allegheny Health Network, Pittsburgh, Pennsylvania
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 1E.9
DOI:  10.1002/9780471729259.mc01e09s40
Online Posting Date:  February, 2016
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Nucleic acids can be obtained in numerous ways from clinical specimens; however, the quality of the nucleic acid is only as good as the sampling and isolation protocol. While nucleic acids may be extracted they may not be representative of the original source. Large areas of tissue and explanted hardware must be successfully surveyed to reflect the overall clinical picture. Once good sampling technique has been established, successful bacterial nucleic acid isolation is essential. Clinical samples may be difficult to process because of the presence of scar tissue, bone, implants, and bacterial biofilms. The following protocols provide details on sampling techniques and DNA isolation from a variety of clinical samples which can then be used in downstream molecular applications including PCR‐MS‐ESI‐TOF technology. © 2016 by John Wiley & Sons, Inc.

Keywords: clinical samples; DNA isolation; nucleic acid; tissue lysis; bacterial lysis; sonication; homogenization

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Clinical Sample Processing and Tissue Lysis: Sonication of Medical Devices
  • Alternate Protocol 1: Clinical Sample Processing and Tissue Lysis: Tissue Samples
  • Alternate Protocol 2: Clinical Sample Processing and Tissue Lysis: Liquid Samples
  • Alternate Protocol 3: Clinical Sample Processing and Tissue Lysis: Swab Samples
  • Basic Protocol 2: Bacterial Lysis and Dna Isolation
  • Basic Protocol 3: DNA Quantification
  • Commentary
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Clinical Sample Processing and Tissue Lysis: Sonication of Medical Devices

  Materials
  • Nuclease‐free water
  • ATL lysis buffer (Qiagen, cat. no. 19076)
  • Proteinase K (Qiagen, cat. no. 19131)
  • Medical device to be processed
  • Plastic bag, sterile
  • Bath ultrasonicator (Branson Ultrasonics, Bransonic CPX952219R)
  • 0.2‐μm, 150‐ml Nalgene Analytical Filter Unit (Thermo Scientific, cat. no. 130‐4020)
  • Vacuum (plumbed or independent unit)
  • Tweezers, sterile
  • Scissors, sterile
  • 50‐ml conical tube, sterile
  • 1.75‐ml microcentrifuge tube, sterile
  • 56°C incubator

Alternate Protocol 1: Clinical Sample Processing and Tissue Lysis: Tissue Samples

  Materials
  • Tissue sample
  • ATL lysis buffer (Qiagen, cat. no. 19076)
  • Proteinase K (Qiagen, cat. no. 19131)
  • 1.75‐ml nuclease‐free microcentrifuge tube, sterile
  • 56°C incubator
  • 50‐ml conical tube, sterile

Alternate Protocol 2: Clinical Sample Processing and Tissue Lysis: Liquid Samples

  Materials
  • Liquid sample (e.g., urine, blood, aspirates, sputum)
  • ATL lysis buffer (Qiagen, cat. no. 19076)
  • Proteinase K (Qiagen, cat. no. 19131)
  • 50‐ml conical tube, sterile
  • Centrifuge
  • Vortex
  • 1.75‐ml microcentrifuge tube, sterile

Alternate Protocol 3: Clinical Sample Processing and Tissue Lysis: Swab Samples

  Materials
  • ATL lysis buffer (Qiagen, cat. no. 19076)
  • Proteinase K (Qiagen, cat. no. 19131)
  • Swab sample
  • 70% ethanol
  • 1.75‐ml microcentrifuge tube, sterile
  • 56°C incubator
  • Tweezers, sterile

Basic Protocol 2: Bacterial Lysis and Dna Isolation

  Materials
  • Sample processed using protocol 1, or Alternate Protocols protocol 21, protocol 32, or protocol 43
  • DNeasy Blood and Tissue Kit (Qiagen, cat. no. 69504)
  • 96% to 100% ethanol
  • 0.1‐mm and 0.7‐mm zirconia beads (BioSpec, cat. nos. 11079101z and 11079107z)
  • TissueLyser II (Qiagen, cat. no. 85300)
  • Centrifuge
  • Heating block for microcentrifuge tubes
  • Microcentrifuge tube cap lock
  • Vortex
CAUTION: Buffer AL contains a chaotropic salt. Take appropriate laboratory safety measures and wear gloves when handling. Chaotropic salts are not compatible with disinfectants containing bleach and can become highly reactive compounds when combined with bleach.

Basic Protocol 3: DNA Quantification

GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
  Costerton, J.W., Lewandowski, Z., Caldwell, D.E., Korber, D.R., and Lappin‐Scott, H.M. 1995. Microbial biofilms. Annu. Rev. Microbiol. 49:711‐745. doi: 10.1146/annurev.mi.49.100195.003431.
  Craig, D.W., Pearson, J.V., Szelinger, S., Sekar, A., Redman, M., Corneveaux, J.J., Pawlowski, T.L., Laub, T., Nunn, G., Stephan, D.A., Homer, N., and Huentelman, M.J. 2008. Identification of genetic variants using bar‐coded multiplexed sequencing. Nat. Methods 5:887‐893. doi: 10.1038/nmeth.1251.
  Ecker, D.J., Drader, J.J., Gutierrez, J., Gutierrez, A., Hannis, J.C., Schink, A., Sampath, R., Blyn, L.B., Eshoo, M.W., Hall, T.A., Tobarmosquera, M., Jiang, Y., Sannes‐Lowery, K.A., Cummins, L.L., Libby, B., Walcott, D.J., Massire, C., Ranken, R., Manalili, S., Ivy, C., Melton, R., Levene, H., Harpin, V., Li, F., White, N., Pear, M., Ecker, J.A., Samant, V., Knize, D., Robbins, D., Rudnick, K., Hajjar, F., and Hofstadler, S.A. 2006. The Ibis T5000 universal biosensor: An automated platform for pathogen identification and strain typing. J. Ass. Lab. Autom. 11:341‐351. doi: 10.1016/j.jala.2006.09.001.
  Esteban, J., Alonso‐Rodriguez, N., del‐Prado, G., Ortiz‐Pérez, A., Molina‐Manso, D., Cordero‐Ampuero, J., and Gómez‐Barrena, E. 2012. PCR‐hybridization after sonication improves diagnosis of implant‐related infection. Acta Orthop. 83:299‐304. doi: 10.3109/17453674.2012.693019.
  Gallagher, S.R. and Desjardins, P.R. 2006. Quantitation of DNA and RNA with Absorption and Fluorescence Spectroscopy. Curr. Protoc. Mol. Biol. 76:3D:A.3D.1‐A.3D.21.
  Jensen, K.H., Dargis, R., Christensen, J.J., and Kemp, M. 2014. Ribosomal PCR and DNA sequencing for detection and identification of bacteria: Experience from 6 years of routine analyses of patient samples. APMIS 122:248‐255. doi: 10.1111/apm.12139.
  Kreft, R., Costerton, J.W., and Ehrlich, G.D. 2013. PCR is changing clinical diagnostics. Microbe 8:15‐20.
  Lleo, M.M., Ghidini, V., Tafi, M.C., Castellani, F., Trento, I., and Boaretti, M. 2014. Detecting the presence of bacterial DNA by PCR can be useful in diagnosing culture‐negative cases of infection, especially in patients with suspected infection and antibiotic therapy. FEMS Microbiol. Lett. 354:153‐160. doi: 10.1111/1574‐6968.12422.
  Trampuz, A., Piper, K.E., Jacobson, M.J., Hanssen, A.D., Unni, K.K., Osmon, D.R., and Patel, R. 2007. Sonication of removed hip and knee prostheses for diagnosis of infection. N. Engl. J. Med. 357:654‐663. doi: 10.1056/NEJMoa061588.
Internet Resources
  http://www.nanodrop.com/ND1/NucleicAcid‐Booklet.html
  Thermo Scientific Nucleic Acid Booklet for the operation of the Thermo Scientific Nanodrop 2000c
  https://www.qiagen.com/us/products/catalog/sample‐technologies/dna‐sample‐technologies/genomic‐dna/dneasy‐blood‐and‐tissue‐kit/#resources
  Qiagen DNeasy Blood and Tissue Kit Handbook
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library