DNA Quality Metrics

Main Findings:

• Acidic solutions will under-represent the 260/280 ratio by 0.2-0.3
• Basic solutions will over-represent the ratio by 0.2-0.3
• Adjusting pH from ~5.4 to 7.5-8.5 significantly increased RNA A260/280 ratios from ~1.5 to 2.0
• pH is critical for accurate nucleic acid quality assessment
• Buffer choice significantly impacts accuracy of purity ratios

Relevance to Course: Demonstrates that DNA quality metrics are influenced by buffer chemistry, teaching students that proper elution buffer selection is critical for accurate quality assessment.

Main Findings:

• Different DNA quality metrics correlate with success of different downstream applications
• 260/280 ratio best predicts PCR success for conventional amplicons
• 260/230 ratio indicates presence of contaminants that may inhibit enzymatic reactions
• DNA integrity (fragment size) is most critical for NGS applications
• Combining multiple quality metrics provides better prediction of application success

Relevance to Course: Provides evidence-based rationale for measuring multiple quality metrics rather than relying on concentration alone.

Main Findings:

• Optimum 260/280 ratio for pure DNA is 1.8, with acceptable purity typically between 1.6 and 2.0
• For pure RNA, the A260/280 ratio is ~2.0
• Ratios appreciably lower (≤1.6) indicate presence of proteins, phenol, or other contaminants
• The 260/230 ratio is used as a secondary measure, with expected values for "pure" DNA between 2.0 and 2.2
• The 260/230 ratio indicates presence of unwanted organic compounds such as Trizol, phenol, guanidine HCl and guanidine thiocyanate

Relevance to Course: Provides clear target values and interpretation guidelines that students can use to assess their own DNA extractions.

Main Findings:

• DNA concentration alone is insufficient to predict PCR success
• 260/280 ratios between 1.7-1.9 correlate with highest PCR success rates
• 260/230 ratios <1.5 strongly predict PCR inhibition
• DNA fragment size >200 bp required for conventional PCR; >500 bp for NGS
• Quality metrics should be evaluated together, not independently

Relevance to Course: Demonstrates why students should measure and interpret all quality metrics, not just concentration, before proceeding to PCR.

Main Findings:

• Absorbance at 260 nm and the 260/280 values are reproducible when low-salt buffer is used as the elution buffer, but not water
• Sample blanking with elution buffer improves accuracy
• Temperature affects absorbance measurements; samples should equilibrate to room temperature
• DNA concentration measurement precision: ±5% for concentrations >10 ng/µL
• Multiple measurements recommended for critical applications

Relevance to Course: Provides practical guidance on proper measurement technique to ensure accurate and reproducible results.

Based on the literature reviewed, comprehensive DNA quality assessment should include:

1. Concentration

• Measure absorbance at 260 nm
• DNA: 1 OD₂₆₀ = 50 µg/mL
• RNA: 1 OD₂₆₀ = 40 µg/mL
• Minimum for PCR: 1-10 ng/µL
• Optimal for PCR: 10-100 ng/µL

2. Purity Ratio 260/280

• 1.8: Pure DNA (optimal)
• 1.6-2.0: Acceptable for most applications
• <1.6: Protein contamination
• >2.0: RNA contamination or alkaline pH
• Contaminants detected: Proteins, phenol, pH issues

3. Purity Ratio 260/230

• 2.0-2.2: Pure DNA (optimal)
• 1.8-2.0: Acceptable for PCR
• <1.8: Organic contamination
• Contaminants detected: Chaotropic salts, phenol, carbohydrates, EDTA

4. DNA Integrity (Fragment Size)

• Methods: Gel electrophoresis, fragment analyzer
• PCR: >200 bp
• Sanger sequencing: >500 bp
• NGS short-read: >500 bp
• NGS long-read: >10 kb

Low 260/280 Ratio (<1.6)

Possible Causes:

• Protein contamination
• Low pH of elution buffer
• Phenol carryover
• Degraded DNA

Solutions:

• Verify pH of elution buffer (should be 7.5-8.5)
• Re-extract with additional proteinase K digestion
• Additional wash steps during extraction
• Ethanol precipitation to remove contaminants

Low 260/230 Ratio (<1.8)

Possible Causes:

• Chaotropic salt carryover (guanidine)
• Ethanol carryover
• EDTA contamination
• Carbohydrate contamination

Solutions:

• Additional wash steps during extraction
• Ensure complete ethanol evaporation
• Clean-up with commercial DNA purification kit
• Dilute DNA 1:5 or 1:10 for PCR

Inconsistent Readings

Possible Causes:

• Bubbles in sample
• Insufficient mixing
• Contaminated measurement surface
• Temperature variation

Solutions:

• Centrifuge briefly before measurement
• Mix by gentle pipetting
• Clean measurement pedestal/cuvette
• Allow samples to equilibrate to room temperature

All citations verified and corrected on: November 5, 2025

Verification method:

• DOIs checked and confirmed to resolve to correct papers using reference verification tools
• Industry guidelines and technical notes reviewed from manufacturer websites
• Main findings cross-referenced across multiple sources
• Quality thresholds validated against current laboratory standards

High-confidence citations:

• Wilfinger et al. (1997) - BioTechniques (foundational paper, widely cited) - DOI verified
• Lucena-Aguilar et al. (2016) - Biopreservation and Biobanking - DOI 10.1089/bio.2015.0064 verified
• Wahlberg et al. (2012) - Biopreservation and Biobanking - DOI 10.1089/bio.2012.0004 verified
• DeNovix Technical Note - Manufacturer documentation verified 2024
• Industry standards - Cross-referenced across BMG LABTECH, Qiagen, Thermo Fisher

Corrections made:

• Fixed Wilfinger header year from (2016) to (1997)
• Replaced Simbolo citation #2 with correct Lucena-Aguilar et al. (2016) citation
• Replaced duplicate citation #4 with Wahlberg et al. (2012) citation
• Updated all DOIs and verified against PubMed/CrossRef databases

Recommendation: These references and quality thresholds are suitable for student handouts and represent current best practices in molecular biology laboratories. Values should be used as guidelines, with institution-specific standards taking precedence.