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Comparison of proteases in DNA extraction via quantitative polymerase chain reaction.

Author(s): Eychner AM, Lebo RJ, Elkins KM

Publication: Anal Biochem, 2015, Vol. 478, Page 128-30

PubMed ID: 25197027 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared DNA yields obtained using four different proteases from nine specimen types (blood, semen, buccal cells, breastmilk, earwax, computer keyboard swabs, chewing gum, and cigarette butts), as well as from diluted blood, semen, and saliva samples. Blood, semen, and breastmilk specimens (50 µl) were pipetted on DNA-free cotton-tipped swabs. Buccal cells and earwax were collected individually on swabs (details not provided). Computer keyboard samples were taken using moistened swabs. Control chewing gum obtained from adult volunteers (chewed for 30 min at least a half-hour after food or drink was consumed) and environmental chewing gum and cigarette butts (collected from the ground at Towson University) were processed whole. DNA extraction was performed using the QIAamp DNA Investigator Kit per manufacturer instructions with the exception of modified kit proteinase K being substituted with unmodified proteinase K, bromelain, or papain. DNA was quantified by real-time qPCR using the Quantifiler TaqMan assay.

   Summary of Findings:

   Amplifiable DNA was obtained from all specimens, regardless of the protease used during extraction. Use of papain and bromelain during extraction resulted in higher average DNA yields (59.1 and 40.2, respectively) from blood than either unmodified proteinase K (32.4 ng) or the QIAamp kit proteinase K (27.8 ng). For semen and buccal cells, use of the kit proteinase K resulted in the highest average DNA yields (74.7 ng and 60.8 ng, respectively) followed by bromelain (50.6 ng and 29.3 ng, respectively), unmodified proteinase K (44.6 ng and 56.6 ng, respectively), and papain (42.9 ng and 43.9 ng, respectively). Similarly, average DNA yields from breastmilk were highest using the kit proteinase K (1.67 ng), but only slightly lower yields were observed when unmodified proteinase K (0.869 ng), bromelain (1.09 ng), or papain (1.20 ng) were used. For earwax, DNA yields were similar among specimens extracted using the different proteases with slightly higher DNA yields obtained when papain was used than with unmodified proteinase K, kit proteinase K, or bromelain (0.848 ng versus 0.690 ng, 0.413 ng, and 0.388 ng; respectively). The average DNA yields were higher when unmodified proteinase K was used for 1:10 and 1:100 diluted blood samples (7.787 and 0.830 ng/µl, respectively), 1:10 and 1:100 diluted semen samples (9.607 and 0.571 ng/µl, respectively), and 1:10 diluted saliva (0.793 ng/µl) than when kit proteinase K, bromelain, or papain were used. The highest DNA yields from control chewing gum were observed when bromelain was used (1.430 ng/µl), from fingerprints on computer keyboards and environmental chewing gum using papain (0.220 ng/µl and 15.697 ng/µl, respectively), and from cigarette butts using the kit proteinase K (4.286 ng/µl).

   Biospecimens

   • Bodily Fluid - Saliva
   • Bodily Fluid - Blood
   • Cell - Buccal
   • Bodily Fluid - Serum
   • Bodily Fluid - Breast Milk
   • Cell - Skin

   Preservative Types

   • None (Fresh)

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   DNA	Real-time qPCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Biospecimen components	Undiluted blood 1:10 diluted blood 1:100 diluted blood Undiluted semen 1:10 diluted semen 1:100 diluted semen 1:10 diluted saliva 1:100 diluted saliva
   Analyte Extraction and Purification	Protein digestion	Unmodified proteinase K Modified kit proteinase K Bromelain Papain

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