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Identification of Streptococcus parasanguinis DNA contamination in human buccal DNA samples.

Mahfuz I, Cheng W, White SJ - BMC Res Notes (2013)

Bottom Line: The use of buccal swabs in clinical and scientific studies is a very popular method of collecting DNA, due to its non-invasive nature of collection.However, contamination of the DNA sample may interfere with analysis.Contamination of buccal-derived DNA with bacterial DNA can be significant, and may influence downstream genetic analysis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Genetic Diseases, Monash Institute of Medical Research, Monash University, 27-31 Wright Street, Clayton 3168, VIC, Australia. stefan.white@monash.edu.

ABSTRACT

Background: The use of buccal swabs in clinical and scientific studies is a very popular method of collecting DNA, due to its non-invasive nature of collection. However, contamination of the DNA sample may interfere with analysis.

Findings: Here we report the finding of Streptococcus parasanguinis bacterial DNA contamination in human buccal DNA samples, which led to preferential amplification of bacterial sequence with PCR primers designed against human sequence.

Conclusion: Contamination of buccal-derived DNA with bacterial DNA can be significant, and may influence downstream genetic analysis. One needs to be aware of possible bacterial contamination when interpreting abnormal findings following PCR amplification of buccal swab DNA samples.

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Related in: MedlinePlus

Alignment of the 153 bp PCR product with the Streptococcus parasanguinis plasmid pFW213 reference sequence. The sequences corresponding to the PCR primers are indicated in bold.
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Figure 3: Alignment of the 153 bp PCR product with the Streptococcus parasanguinis plasmid pFW213 reference sequence. The sequences corresponding to the PCR primers are indicated in bold.

Mentions: Contamination was detected during mutation screening of the Desmoplakin (DSP) gene in buccal DNA of Bladder Exstrophy Epispadias Complex (BEEC) patients. Use of samples for research was approved by the Research Ethics Committee of Royal Children’s Hospital (Approval Number# HREC28140A), Melbourne, Australia. Informed consent was also obtained from the patients or parents/guardians. The intention was to screen DSP exon 4 for potential sequence variants using High Resolution Melting (HRM) and Sanger Sequencing. For this, we designed exon-specific forward (5′ CTGTTTTCCTGCAGTGGTT 3′) and reverse (5′ TGGCCTGCACAGGTTTG 3′) primers, predicted to generate a 254 bp product. HRM was performed on 22 samples as previously described [11]. Five samples gave aberrant curves with HRM (Figure 1), and agarose gel analysis showed the presence of two bands in matching samples (Figure 2). Sanger sequence analysis showed that the 153 bp fragment did not align with any human sequence. Alignment with other organisms showed 98% identity with Streptococcus parasanguinis plasmid pFW213 [GenBank:EU685104.1] (Figure 3).


Identification of Streptococcus parasanguinis DNA contamination in human buccal DNA samples.

Mahfuz I, Cheng W, White SJ - BMC Res Notes (2013)

Alignment of the 153 bp PCR product with the Streptococcus parasanguinis plasmid pFW213 reference sequence. The sequences corresponding to the PCR primers are indicated in bold.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4222080&req=5

Figure 3: Alignment of the 153 bp PCR product with the Streptococcus parasanguinis plasmid pFW213 reference sequence. The sequences corresponding to the PCR primers are indicated in bold.
Mentions: Contamination was detected during mutation screening of the Desmoplakin (DSP) gene in buccal DNA of Bladder Exstrophy Epispadias Complex (BEEC) patients. Use of samples for research was approved by the Research Ethics Committee of Royal Children’s Hospital (Approval Number# HREC28140A), Melbourne, Australia. Informed consent was also obtained from the patients or parents/guardians. The intention was to screen DSP exon 4 for potential sequence variants using High Resolution Melting (HRM) and Sanger Sequencing. For this, we designed exon-specific forward (5′ CTGTTTTCCTGCAGTGGTT 3′) and reverse (5′ TGGCCTGCACAGGTTTG 3′) primers, predicted to generate a 254 bp product. HRM was performed on 22 samples as previously described [11]. Five samples gave aberrant curves with HRM (Figure 1), and agarose gel analysis showed the presence of two bands in matching samples (Figure 2). Sanger sequence analysis showed that the 153 bp fragment did not align with any human sequence. Alignment with other organisms showed 98% identity with Streptococcus parasanguinis plasmid pFW213 [GenBank:EU685104.1] (Figure 3).

Bottom Line: The use of buccal swabs in clinical and scientific studies is a very popular method of collecting DNA, due to its non-invasive nature of collection.However, contamination of the DNA sample may interfere with analysis.Contamination of buccal-derived DNA with bacterial DNA can be significant, and may influence downstream genetic analysis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Genetic Diseases, Monash Institute of Medical Research, Monash University, 27-31 Wright Street, Clayton 3168, VIC, Australia. stefan.white@monash.edu.

ABSTRACT

Background: The use of buccal swabs in clinical and scientific studies is a very popular method of collecting DNA, due to its non-invasive nature of collection. However, contamination of the DNA sample may interfere with analysis.

Findings: Here we report the finding of Streptococcus parasanguinis bacterial DNA contamination in human buccal DNA samples, which led to preferential amplification of bacterial sequence with PCR primers designed against human sequence.

Conclusion: Contamination of buccal-derived DNA with bacterial DNA can be significant, and may influence downstream genetic analysis. One needs to be aware of possible bacterial contamination when interpreting abnormal findings following PCR amplification of buccal swab DNA samples.

Show MeSH
Related in: MedlinePlus