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Genotyping for DQA1 and PM loci in urine using PCR-based amplification: effects of sample volume, storage temperature, preservatives, and aging on DNA extraction and typing.

Author(s): Vu NT, Chaturvedi AK, Canfield DV

Publication: Forensic Sci Int, 1999, Vol. 102, Page 23-34

PubMed ID: 10423850 PubMed Review Paper? No

Purpose of Paper

This paper investigated the effects of gender, storage conditions, sample volume, concentration methods, DNA extraction methods, and the use of chemical preservatives on the quantity of DNA recovered and the ability to successfully perform PCR-based genotyping on urine specimens. DQA1 and PM amplification were performed which targeted the following genetic loci: HLA, LDLR, GYPA, HBGG, D7S8, and GC.

Conclusion of Paper

Urine specimens obtained from females yielded more DNA than samples from males and subsequently resulted in higher successful DNA typing rates. For specimens obtained from males, freezing and storage at room temperature (RT) negatively impacted the amount of DNA recovered and the typeable frequency. This effect increased with storage duration and was less severe at RT than -20 degrees C. In females, storage conditions had no significant impact. Higher initial specimen volumes yielded more DNA but did not impact typing results. Purification by dialfiltration did not improve DNA recovery or successful typing rates as compared with concentration by centrifugation or micro-centrifugation. No samples were typeable when 1% sodium fluoride was added as a preservative. However, adding 0.25% sodium azide to urine specimens resulted in a high rate of DNA typing after storage at 4 degrees C for up to 20 days.

Studies

Study Purpose

The purpose of this study was to examine the effects of different concentration techniques on the ability to type DNA recovered from either fresh male urine samples or those that had been frozen for 24 hours.

Summary of Findings:

Typeable blots obtained from samples subjected to dialfiltration exhibited a weaker color reaction than those from samples obtained after centrifugation and micro-centrifugation. Therefore the two simpler latter techniques were used in subsequent experiments. DNA from fresh samples, rather than those frozen for 24 hours was better suited for genotyping.

Biospecimens

Bodily Fluid - Urine

Preservative Types

Frozen
None (Fresh)

Diagnoses:

Normal

Platform:

Analyte	Technology Platform
DNA	Dot blot or slot blot

Pre-analytical Factors:

Classification	Pre-analytical Factor	Value(s)
Analyte Extraction and Purification	Analyte purification	Centrifugation Micro-centrifugation Dialfiltration
Biospecimen Aliquots and Components	Aliquot size/volume	1 ml 2 ml 5 ml
Biospecimen Preservation	Type of fixation/preservation	None (fresh) Frozen
Preaquisition	Patient gender	Male
Storage	Storage duration	0 h 24 h
Storage	Storage temperature	-20 degrees C

Study Purpose

The purpose of this study was to compare the efficiency of organic extraction (chloroform: phenol: isoamyl alcohol) with Chelex extraction (using Chelex 100 resin) of DNA from 6 male urine specimens by determining whether the samples could be successfully typed. Both fresh specimens and those that had been frozen for 7 days were analyzed.

Summary of Findings:

DNA extracted using both organic methods and Chelex resin was able to be typed (6/6 samples from fresh specimens for each method). However, when specimens were frozen for 7 days, only 4/6 samples were successfully typed for each extraction method. Although typeable results were obtained with organically extracted DNA, the signals on the dot blot were less intense so the Chelex method was used for further studies.

Biospecimens

Bodily Fluid - Urine

Preservative Types

Frozen
None (Fresh)

Diagnoses:

Normal

Platform:

Analyte	Technology Platform
DNA	Dot blot or slot blot

Pre-analytical Factors:

Classification	Pre-analytical Factor	Value(s)
Preaquisition	Patient gender	Male
Analyte Extraction and Purification	Analyte purification	Micro-centrifuge
Biospecimen Aliquots and Components	Aliquot size/volume	1 ml
Biospecimen Preservation	Type of fixation/preservation	None (fresh) Frozen
Storage	Storage temperature	-20 degrees C
Storage	Storage duration	0 d 7 d
Analyte Extraction and Purification	Analyte isolation method	Organic extraction Chelex extraction

Study Purpose

The purpose of this study was to determine the effects of gender, sample volume, storage temperature, and storage duration on the quantity of DNA able to be extracted from urine specimens and its ability to be used for genotyping.

Summary of Findings:

Significantly larger amounts of DNA were obtained from female specimens than from male specimens however, the results were still variable among the 13 female subjects included in the study. Centrifugation, as opposed to micro-centrifugation, resulted in a slightly higher yield of DNA. The authors postulate this was due to the larger starting sample volume (5 ml). Both frozen storage and storage at RT significantly decreased the yield of DNA after 24 hours, regardless of gender. These effects were more pronounced with frozen specimens. Interestingly, while storage temperature and duration had similar negative effects on the typeability of male urine specimens, the typeability of female urine specimens was relatively unaffected.

Biospecimens

Bodily Fluid - Urine

Preservative Types

Frozen
None (Fresh)

Diagnoses:

Normal

Platform:

Analyte	Technology Platform
DNA	Chemiluminescence
DNA	Dot blot or slot blot

Pre-analytical Factors:

Classification	Pre-analytical Factor	Value(s)
Preaquisition	Patient gender	Female Male
Analyte Extraction and Purification	Analyte purification	Centrifugation Micro-centrifugation
Biospecimen Aliquots and Components	Aliquot size/volume	1 ml 5 ml
Biospecimen Preservation	Type of fixation/preservation	None (fresh) Frozen
Storage	Storage temperature	-20 degrees C RT
Storage	Storage duration	0 d 1 d 2 d 7 d
Analyte Extraction and Purification	Analyte isolation method	Chelex extraction

Study Purpose

The purpose of this study was to examine the effects of preservatives on 6 male urine specimens by determining whether extracted DNA could be successfully typed. Both fresh specimens and those that had been refrigerated or frozen for up to 30 days were analyzed.

Summary of Findings:

The addition of 1% sodium fluoride to urine specimens completely blocked the typeability of extracted DNA regardless of storage temperature or duration. However, when 0.25% sodium azide was added, all DNA extracted from fresh specimens (6/6), as well as those that were refrigerated for up to 20 days (6/6), was able to be typed. Only half of the samples from frozen storage were typeable after 20 days (3/6) and longer refrigeration or frozen storage (30 d) decreased the number of typeable results (1/6).

Biospecimens

Bodily Fluid - Urine

Preservative Types

Frozen
Other Preservative
None (Fresh)

Diagnoses:

Normal

Platform:

Analyte	Technology Platform
DNA	Dot blot or slot blot

Pre-analytical Factors:

Classification	Pre-analytical Factor	Value(s)
Preaquisition	Patient gender	Male
Analyte Extraction and Purification	Analyte purification	Micro-centrifugation
Biospecimen Aliquots and Components	Aliquot size/volume	1 ml
Biospecimen Preservation	Type of fixation/preservation	None (fresh) Frozen Refrigeration
Biospecimen Preservation	Fixative additive/buffer	Sodium azide Sodium fluoride
Storage	Storage temperature	-20 degrees C 4 degrees C RT
Storage	Storage duration	0 d 7 d 20 d 30 d
Analyte Extraction and Purification	Analyte isolation method	Chelex extraction

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