Collection of buccal cell DNA using treated cards.

Author(s): Harty LC, Garcia-Closas M, Rothman N, Reid YA, Tucker MA, Hartge P

Publication: Cancer Epidemiol Biomarkers Prev, 2000, Vol. 9, Page 501-6

PubMed ID: 10815695 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the effects of patient condition, collection parameters, storage and PCR amplification protocol on DNA yield and PCR success from buccal cells. Buccal cells were collected by cytobrush, transferred to an IsoCode collection card, and stored at room temperature with dessicant for 5 days prior to start of experimental storage.

   Summary of Findings:

   Compared to fresh specimens, the DNA yields were 45.2% and 57.2% lower when specimens were stored for 9 months at room temperature (p=0.01) or -70 degrees C (p=0.02), respectively. However, when specimens were stored at -20 degrees C, a non-significant 29.6% decrease was observed in DNA yields compared to those from fresh specimens. Saliva from the single patient with a chronic dry mouth and the three smokers yielded less DNA than other patients. The average DNA yield from the portion of the card to which the saliva was directly applied was slightly higher than in the adjacent area to which it spread (137.5 ng versus 121 ng). The 268, 536 and 989 bp fragments of beta-globin were successfully amplified in 98.1%, 96.1% and 90.4% of cases. Failure to amplify these fragments was associated with a The specimens which failed to amplify the 989 bp fragment also failed to amplify the 268 and 536 bp fragments lower DNA yield. When DNA was stored for 1 week at -20 degrees C after being previously kept for 2 months at room temperature, a 536 bp product was amplified in 88.5% of specimens compared to 96.1% of specimens where DNA samples were not frozen after the initial room temperature storage. 5 of the 6 frozen DNA samples that failed amplification attempts using standard Taq polymerase were subsequently amplified when AmpliTaq Gold was used.

   Biospecimens

   • Cell - Buccal

   Preservative Types

   • None (Fresh)
   • Frozen

   Diagnoses:

   • Normal
   • Other diagnoses

   Platform:

   Analyte	Technology Platform
   DNA	PCR
   DNA	Southern blot

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Storage temperature	-70 degrees C -20 degrees C Room temperature
   Storage	Storage duration	1 week 90 days
   Biospecimen Preservation	Type of fixation/preservation	Frozen None (fresh)
   PCR Specific	Length of gene fragment	268 bp 536 bp 989 bp
   PCR Specific	Targeted nucleic acid	Beta-globin
   PCR Specific	Nucleic acid amplification	AmpliTaq Gold Taq polymerase
   Preaquisition	Other drugs	Smoker Non-smoker
   Preaquisition	Diagnosis/ patient condition	Normal Dry mouth
   Biospecimen Aliquots and Components	Biospecimen heterogeneity	Biospecimen core Biospecimen periphery

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