How to Choose DNA Extraction Method

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How to Choose DNA Extraction Method

Decision Guide for Column-Based vs. Magnetic Bead Extraction

ENTM201L - Theory Supplement

Overview

DNA extraction is not a one-size-fits-all procedure. The choice between column-based extraction and magnetic bead extraction depends on your downstream application, sample type, throughput needs, and budget. This guide helps you make informed decisions about which method to use for your mosquito DNA extraction.

Two Methods Compared:

• Column-based extraction (Zymo Quick-DNA Tissue/Insect Microprep Kit)
• Magnetic bead extraction (BioDynami Genomic DNA Extraction Kit)

Both methods work well for mosquito specimens, but they excel in different applications.

Quick Decision Guide

Use Column-Based Extraction When:

• Your downstream application is PCR or Sanger sequencing
• You need results quickly (15-20 minute protocol)
• You are processing many samples (scalable to 96-well format)
• DNA fragment size up to 40 kb is sufficient
• Budget allows moderate per-sample cost

Use Magnetic Bead Extraction When:

• Your downstream application is long-read sequencing (PacBio, Nanopore)
• You need high molecular weight DNA (50-150 kb)
• You want maximum DNA yield from limited samples
• You are doing genome assembly or structural variant analysis
• You want lower per-sample cost
• You can afford longer processing time (2 hours)

Understanding High Molecular Weight (HMW) DNA

What is HMW DNA?

High Molecular Weight DNA refers to DNA fragments that are very long and intact:

• HMW DNA: Typically >40 kb (40,000 base pairs)
• Ultra-HMW DNA: >100 kb (100,000 base pairs)
• Standard genomic DNA: 10-40 kb

Why HMW DNA Matters

For short-read applications (PCR, Sanger sequencing):

• Fragment size is NOT critical
• PCR amplicons are typically <5 kb
• Fragmented DNA still contains intact target sequences
• Both column and magnetic bead methods work equally well

For long-read applications (PacBio, Nanopore):

• Fragment size is CRITICAL
• Long-read sequencers require >40 kb fragments
• Fragmented DNA produces poor quality assemblies
• Only magnetic bead methods preserve HMW DNA

How Magnetic Beads Preserve HMW DNA

Magnetic bead extraction minimizes mechanical stress on DNA:

Minimal shear forces:

• No centrifugation through membranes (unlike columns)
• DNA remains in solution throughout process
• Gentle magnetic separation instead of high-speed spinning
• Tapping/gentle mixing instead of vigorous vortexing

Why columns fragment DNA:

• High centrifugal force (10,000-14,000 x g)
• DNA forced through small silica pores (~5 µm)
• Shear stress during binding and washing steps
• Multiple spin cycles compound fragmentation

Result:

• Magnetic beads: DNA fragments 50-150 kb
• Columns: DNA fragments 10-50 kb

When HMW DNA is Essential

Long-read sequencing platforms:

• PacBio (Pacific Biosciences): Requires >40 kb, optimal >100 kb
• Oxford Nanopore: Requires >20 kb, optimal >50 kb
• 10x Genomics: Requires >40 kb for linked-read sequencing

Specific applications:

• Genome assembly: Longer reads = fewer gaps, better contiguity
• Structural variants: Detect large insertions, deletions, inversions
• Haplotype phasing: Link distant variants on same chromosome
• Repeat regions: Span repetitive elements that confuse short reads

Example:

If you are sequencing the mosquito genome for the first time, you need HMW DNA. A mosquito genome is ~200 million base pairs with many repetitive regions. Short fragments (10-40 kb from columns) would create a fragmented assembly with thousands of gaps. Long fragments (50-150 kb from magnetic beads) span repeats and produce a more contiguous assembly.

Comprehensive Method Comparison

Side-by-Side Feature Comparison

Feature	Magnetic Beads (BioDynami)	Column (Zymo Quick-DNA)
Binding Principle	DNA binds reversibly to magnetic particles in high salt	DNA binds silica membrane in chaotropic conditions
Separation Method	Magnetic rack (physical separation)	Centrifugation (mechanical separation)
Lysis Method	Manual grinding + Proteinase K digestion	Bead-beating with Disruptor Genie
Protocol Time	120 minutes (2 hours)	30 minutes (15-20 min hands-on)
Hands-on Steps	8-10 pipetting steps	5-6 pipetting steps
Mechanical Stress	Minimal (no centrifugation)	High (14,000 x g spin through membrane)
Expected Fragment Size	50-150 kb (HMW DNA)	10-50 kb (genomic DNA)
Yield from 1 Mosquito	1.5-2 ng/µL in 50 µL = 75-100 ng total	10-20 ng/µL in 50 µL = 0.5-1 µg total
A260/A280 Ratio	1.80-2.0 (excellent purity)	1.75-1.90 (good purity)
A260/A230 Ratio	2.0-2.2 (minimal salt contamination)	1.8-2.0 (slight salt carryover)
Cost per Sample	~$2-3 (lower)	~$4-5 (higher)
Automation Potential	Easy (magnetic robots available)	Difficult (requires centrifuge integration)
Throughput	Limited to magnetic rack capacity (12-24 samples)	Scalable to 96-well format (high-throughput)
Storage Stability	Requires careful mixing before use	Ready-to-use columns
Best Applications	Long-read sequencing, genome assembly, structural variants	PCR, Sanger sequencing, SNP genotyping, barcoding

Speed Comparison

Magnetic Bead Extraction (Total: ~120 minutes):

1. Sample preparation: 5 minutes

2. Tissue lysis (56°C incubation): 45 minutes

3. DNA binding to beads: 20 minutes

4. Magnetic separation and washing (3 washes): 30 minutes

5. DNA elution: 15 minutes

6. Quality assessment: 15 minutes

Column Extraction (Total: ~30 minutes):

1. Sample preparation: 2 minutes

2. Bead-beating lysis: 10 minutes (automated)

3. Centrifugation and clarification: 5 minutes

4. DNA binding to column (2 rounds): 5 minutes

5. Washing and elution: 8 minutes

Winner for Speed: Column extraction (4x faster)

Cost Comparison

Magnetic Bead Kit (~$2-3 per sample):

• Reagents are less expensive per extraction
• Can be scaled down for single mosquito
• Beads can sometimes be regenerated (cost savings)
• Requires magnetic rack (one-time equipment cost: ~$50-100)

Column Kit (~$4-5 per sample):

• Pre-packaged columns are convenient but more expensive
• Fixed reagent volumes (less flexible)
• Requires Disruptor Genie (one-time equipment cost: ~$1,000)

Winner for Cost: Magnetic beads (for small-scale)

DNA Quality Comparison

Purity (A260/A280 ratio):

• Magnetic beads: 1.80-2.0 (excellent)
• Columns: 1.75-1.90 (good, acceptable for mosquito DNA)
• Why difference? Magnetic beads have more wash steps; columns may retain trace proteins from rapid processing

Salt Contamination (A260/A230 ratio):

• Magnetic beads: 2.0-2.2 (minimal contamination)
• Columns: 1.8-2.0 (slight salt carryover)
• Why difference? Silica membranes can leach silicates; magnetic beads release DNA cleanly in low-salt buffer

Integrity (Fragment Size):

• Magnetic beads: 50-150 kb (HMW DNA, visible as sharp high band on gel)
• Columns: 10-50 kb (genomic DNA, visible as broad smear 10-40 kb on gel)
• Why difference? Magnetic separation avoids shear forces from centrifugation through membranes

Winner for Quality: Magnetic beads (higher purity, HMW DNA)

Yield Comparison by Preservation Method

DNA yield varies depending on how mosquito specimens were preserved:

Preservation Method	Magnetic Beads (ng/µL)	Column (ng/µL)	Difference
-80°C Frozen	150-200	120-180	Beads ~15% higher
-20°C Frozen	100-150	80-120	Beads ~20% higher
95% Ethanol	80-120	60-100	Beads ~20% higher
70% Ethanol	40-80	30-60	Beads ~25% higher
Silica Gel Dried	30-60	20-40	Beads ~30% higher

Why magnetic beads yield more:

• DNA binds reversibly to beads (less permanent loss)
• No membrane saturation limit (columns have binding capacity ~5 µg)
• Less DNA trapped in matrix
• Better recovery of degraded DNA (short fragments bind beads but might wash through columns)

Winner for Yield: Magnetic beads (consistently 15-30% higher)

Application-Specific Recommendations

For Standard PCR (<5 kb Amplicons)

Example: COI barcoding (712 bp amplicon) Requirements:

• DNA concentration: 10-50 ng/µL
• A260/A280: >1.7 (slight protein contamination tolerated)
• A260/A230: >1.8 (salt contamination more problematic for enzyme activity)
• Fragment size: Not critical (fragmented DNA contains intact COI targets)

Recommended Method: Either method works equally well

• Column extraction: Faster, higher throughput
• Magnetic beads: Better purity, higher yield

Decision factors:

• If processing many samples (>20): Use columns (96-well format)
• If processing few samples (<10): Use magnetic beads (better yield per sample)
• If time is limited: Use columns (30 min vs. 2 hours)
• If budget is limited: Use magnetic beads (lower cost per sample)

For Sanger Sequencing (Post-PCR Cleanup)

Requirements:

• PCR product concentration: 20-50 ng/µL
• A260/A280: >1.8 (proteins interfere with sequencing polymerase)
• A260/A230: >2.0 (salts interfere with capillary electrophoresis)
• Single band on gel (no primer dimers or non-specific products)

Recommended Method: Either method produces suitable template

• Initial DNA extraction quality affects PCR success
• Both methods produce DNA compatible with downstream PCR

Post-PCR cleanup:

• Use column cleanup (Zymo DNA Clean & Concentrator) or
• Use enzymatic cleanup (ExoSAP-IT) if PCR product is very clean

For Long-Range PCR (>5 kb Amplicons)

Example: Amplifying entire mitochondrial genome (15-20 kb) Requirements:

• DNA concentration: 50-100 ng/µL (higher than standard PCR)
• A260/A280: >1.8 (strict requirement)
• A260/A230: >2.0 (strict requirement)
• Fragment size: CRITICAL - template must be >20 kb for efficient amplification

Recommended Method: Magnetic bead extraction (essential)

• HMW DNA (50-150 kb) spans entire target region
• Column DNA (10-50 kb) may not contain intact long templates
• Success rate dramatically higher with HMW template

Why HMW matters for long-range PCR:

If your template DNA is fragmented to 10-20 kb pieces, and you need to amplify a 15 kb region, statistically many template molecules will have breaks within the target region. PCR fails when template is fragmented within the amplicon. HMW DNA (>50 kb) ensures intact templates.

For PacBio Long-Read Sequencing

Requirements:

• DNA concentration: >200 ng/µL (high concentration essential)
• A260/A280: 1.8-2.0 (strict)
• A260/A230: 2.0-2.2 (strict)
• Fragment size: >40 kb required, >100 kb optimal
• DNA Integrity Number (DIN): >8.0 (requires Bioanalyzer)

Recommended Method: Magnetic bead extraction (ONLY option)

• Column DNA is too fragmented (<50 kb) for PacBio
• PacBio library prep requires ultra-HMW DNA
• Even slight fragmentation reduces read lengths

PacBio Applications:

• De novo genome assembly (mosquito genome sequencing)
• Full-length transcript sequencing (isoform analysis)
• Structural variant detection (large insertions/deletions)
• Haplotype phasing (long-range linkage)

For Oxford Nanopore Sequencing

Requirements:

• DNA concentration: >100 ng/µL
• A260/A280: 1.8-2.0
• A260/A230: 2.0-2.2
• Fragment size: >20 kb minimum, >50 kb optimal

Recommended Method: Magnetic bead extraction (preferred)

• Column DNA (10-50 kb) produces shorter Nanopore reads
• Magnetic bead DNA (50-150 kb) produces longer reads (better assemblies)
• Nanopore is more tolerant of slightly fragmented DNA than PacBio

Nanopore Applications:

• Rapid species identification (real-time sequencing)
• Portable field sequencing (MinION device)
• Amplicon sequencing for resistance genes
• Metagenomics (pathogen detection in mosquito pools)

For Illumina Short-Read Sequencing

Requirements:

• DNA concentration: 20-100 ng/µL
• A260/A280: 1.8-2.0
• A260/A230: 2.0-2.2
• Fragment size: Not critical (Illumina fragments DNA during library prep anyway)

Recommended Method: Either method works

• Column extraction: Faster, suitable for routine Illumina prep
• Magnetic beads: Better purity, preferred for low-input samples

Note: Illumina library prep includes enzymatic fragmentation (sonication or Tn5 tagmentation) that cuts DNA to 300-500 bp fragments. Starting with 10 kb or 100 kb DNA makes no difference for final library quality.

For Genome Assembly Projects

Requirements:

• HMW DNA is essential
• Typical approach: Hybrid assembly combining short and long reads
• Short reads (Illumina): High accuracy, use either extraction method
• Long reads (PacBio/Nanopore): Scaffolding, requires magnetic bead extraction

Recommended Method: Magnetic bead extraction for long-read fraction

• Extract multiple mosquitoes using magnetic beads
• Pool DNA to achieve >5 µg total yield
• Use portion for Illumina (can fragment), portion for PacBio (keep HMW)

For SNP Genotyping and Microsatellite Analysis

Requirements:

• DNA concentration: 10-50 ng/µL
• A260/A280: >1.8
• A260/A230: >1.8
• Fragment size: Not critical (target loci are small)

Recommended Method: Column extraction (preferred for throughput)

• Population genetics studies require processing hundreds of samples
• 96-well column format enables high-throughput extraction
• Automated liquid handling compatible with column workflows

For Insecticide Resistance Genotyping

Requirements:

• DNA from individual mosquitoes (not pooled)
• High-throughput processing (surveillance programs test hundreds)
• Targets are PCR-based (kdr, ace-1, esterases)

Recommended Method: Column extraction (industry standard)

• Fast protocol (can process 96 samples in 2 hours)
• Sufficient quality for PCR-based genotyping
• Cost-effective for large-scale surveillance

Decision Flowchart (Interactive Wizard)

Scientific Reasoning: WHY Choose One Over the Other?

Molecular Mechanisms Underlying Method Differences

DNA Binding Chemistry

Magnetic Beads:

• Beads coated with carboxyl groups (-COO⁻)
• In high salt (Binding Buffer), DNA backbone phosphates interact with bead surface
• Binding is reversible - DNA releases in low-salt Elution Buffer
• Mechanism: Electrostatic interaction modulated by ionic strength

Silica Columns:

• Silica (SiO₂) membrane with silanol groups (Si-OH)
• Chaotropic salts (guanidinium) disrupt water structure around DNA
• DNA binds silica through hydrogen bonding between phosphate and silanol
• Elution requires low-salt conditions or slightly alkaline pH (DNA deprotonates, loses affinity)

Implication: Both methods rely on salt-mediated binding, but magnetic beads preserve DNA structure better because binding/release involves less chemical stress.

Shear Forces and DNA Fragmentation

Physics of DNA Shearing:

• DNA is a long, flexible polymer
• Shear stress occurs when fluid flows past DNA at different velocities
• Formula: Shear rate (γ) = velocity gradient = ΔV/Δy
• High shear rates break DNA at weakest points (A-T rich regions, nicks from DNases)

Column Extraction Shear Forces:

• Centrifugation at 10,000-14,000 x g
• Liquid forced through 5 µm pores at high velocity
• Creates turbulent flow and velocity gradients
• Shear rate: ~10,000 s⁻¹ (enough to fragment DNA >50 kb)

Magnetic Bead Separation:

• No centrifugation (beads pulled gently by magnet)
• DNA remains in solution (laminar flow during mixing)
• Shear rate: ~100 s⁻¹ (100x lower than columns)
• Result: DNA fragments remain >50 kb

Mathematical Insight:

DNA contour length for 100 kb = 100,000 bp × 0.34 nm/bp = 34 µm

When forced through a 5 µm pore, a 34 µm DNA molecule must fold/compress. If flow is too fast, DNA breaks rather than deforming. Magnetic separation avoids this entirely.

Proteinase K Digestion vs. Bead-Beating

Magnetic Bead Protocol (Proteinase K):

• Proteinase K digests proteins at 56°C for 30-45 minutes
• Slow, gentle enzymatic lysis
• Preserves DNA integrity (no mechanical stress)
• Complete protein removal (improves purity)

Column Protocol (Bead-Beating):

• Mechanical disruption with ceramic beads
• Very fast (5-10 minutes)
• Physical shearing of cells AND DNA
• Trade-off: Speed vs. HMW DNA preservation

When does this matter?

• For PCR: Doesn't matter (both methods provide sufficient template)
• For long-read sequencing: Critical (Proteinase K preserves HMW, bead-beating fragments)

Troubleshooting: Which Method to Use When Standard Protocol Fails?

Scenario 1: Low DNA Yield from Column Extraction

Problem: NanoDrop reads 8 ng/µL (below 50 ng/µL expected) Possible Causes:

• Incomplete lysis (bead-beating time too short)
• DNA loss during washing (excessive wash buffer volume)
• Poor elution (elution buffer not pre-warmed)

Solutions:

1. Increase bead-beating time from 10 min to 15 min

2. Reduce wash buffer volume from 500 µL to 300 µL

3. Pre-warm elution buffer to 65°C before use

4. Try magnetic bead extraction instead - inherently higher yield

When to switch methods:

If yield is consistently <10 ng/µL with columns, switch to magnetic beads (better for low-input samples).

Scenario 2: Poor Purity Ratios (Low A260/A280)

Problem: A260/A280 = 1.5 (indicates protein contamination) Column Extraction Solutions:

• Add extra wash step with g-DNA Wash Buffer
• Increase bead-beating time (ensure complete lysis)
• Use freshly prepared lysis buffer

Magnetic Bead Solutions:

• Extend Proteinase K incubation to 60 minutes
• Add extra wash with Wash Buffer 2
• Ensure beads are fully resuspended during washing

Which method handles protein contamination better?

Magnetic beads - multiple wash steps and Proteinase K digestion are more effective than rapid column washes.

Scenario 3: PCR Inhibition Despite Adequate DNA Concentration

Problem: Qubit reads 50 ng/µL, but PCR fails (no product) Likely Cause: PCR inhibitors present despite good concentration Common Inhibitors:

• Chaotropic salts (from binding buffers)
• Ethanol (from wash buffers)
• Melanin (from mosquito eyes)
• Heme (from blood-fed mosquitoes)

Solutions:

1. Dilute template 1:5 (dilutes inhibitors more than DNA)

2. Add BSA to PCR (binds inhibitors)

3. Re-extract with additional wash steps

4. Try alternative method:

- If column failed → Try magnetic beads (cleaner elution)

- If magnetic beads failed → Try column (different buffer chemistry may avoid inhibitor carryover)

Scenario 4: Need HMW DNA but Only Have Column Kit

Problem: You need >40 kb DNA for PacBio, but only have column extraction kit Mitigation Strategies:

1. Minimize centrifugation force:

- Reduce speed from 14,000 x g to 8,000 x g (slower but gentler)

- Increases protocol time but reduces shear

2. Minimize number of spin cycles:

- Combine wash steps if possible

- Skip optional washes

3. Handle DNA gently post-extraction:

- No vortexing (tap to mix)

- Use wide-bore pipette tips

- Avoid freeze-thaw cycles

Reality Check:

Even with optimizations, column DNA rarely exceeds 50 kb. For true HMW DNA (>100 kb), magnetic bead extraction (or phenol-chloroform) is necessary.

Cost-Benefit Analysis

Time Investment

If you value time:

• Column extraction is 4x faster (30 min vs. 120 min)
• For 10 samples: Columns = 1.5 hours, Beads = 3-4 hours
• For 96 samples: Columns = 2 hours (parallel processing), Beads = not practical

If you value DNA quality over time:

• Magnetic beads provide better purity and HMW DNA
• Worth the extra 90 minutes for critical applications

Financial Investment

Budget Calculation (per sample):

Column extraction:

• Kit cost: ~$5/sample
• Tips and tubes: ~$0.50/sample
• Total: ~$5.50/sample

Magnetic bead extraction:

• Kit cost: ~$3/sample
• Tips and tubes: ~$0.50/sample
• Total: ~$3.50/sample

For 100 samples:

• Columns: 100 × $5.50 = $550
• Magnetic beads: 100 × $3.50 = $350
• Savings with beads: $200 (36% cheaper)

Equipment Costs (one-time):

• Magnetic rack: ~$50-100
• Disruptor Genie: ~$1,000
• If you don't have Disruptor Genie, magnetic beads are more cost-effective

Opportunity Cost

What if your extraction fails?

• Column failure: Fast to re-extract (30 min lost)
• Magnetic bead failure: Longer to re-extract (2 hours lost)

What if you extract with wrong method?

• Used columns but need HMW DNA: Must re-extract with beads (sample consumed, time/money lost)
• Used beads but only need PCR template: No problem (over-qualified DNA works fine)

Risk-Averse Strategy: If uncertain, use magnetic beads (provides maximum flexibility for downstream applications).

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Real-World Case Studies

Case Study 1: Mosquito DNA Barcoding Project (100 Samples)

Goal: Species identification of mosquito surveillance samples using COI barcoding Requirements:

• PCR amplification of 712 bp COI gene
• Sanger sequencing
• Process 100 samples per week

Method Chosen: Column extraction (96-well format) Reasoning:

• High throughput needed (100 samples/week)
• COI barcoding doesn't require HMW DNA
• 96-well format enables parallel processing
• Total time: 2 hours for 96 samples (vs. impossible with magnetic beads)
• Cost: $550 for 100 samples (acceptable for surveillance budget)

Outcome:

• PCR success rate: 98% (2 failures due to degraded samples)
• Sanger sequencing success: 96% (4 failures due to low PCR yield)
• Overall success: Excellent for intended application

Lesson: When throughput is priority and HMW DNA is not needed, columns are superior.

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Case Study 2: Aedes aegypti Genome Sequencing Project

Goal: Assemble chromosome-level reference genome for local mosquito population Requirements:

• PacBio long-read sequencing (20-50 kb reads)
• Illumina short-read sequencing (150 bp paired-end)
• Structural variant detection

Method Chosen: Magnetic bead extraction Reasoning:

• PacBio requires HMW DNA (>40 kb, ideally >100 kb)
• Column DNA (10-50 kb) insufficient for PacBio library prep
• Willing to invest extra time (2 hours) for HMW DNA
• Only need 10-20 mosquitoes (low throughput acceptable)

Protocol Modifications:

• Extracted 50 mosquitoes individually using magnetic beads
• Quantified each extraction (selected top 20 with >150 ng/µL)
• Pooled 20 samples to achieve 10 µg total HMW DNA
• Used portion for PacBio library (~5 µg)
• Used portion for Illumina library (~1 µg, fragmented during library prep)

Outcome:

• PacBio N50 read length: 35 kb (excellent)
• Genome assembly N50: 2.5 Mb (chromosome-scale scaffolds)
• Detected 1,247 structural variants not visible in previous assemblies
• Overall success: Would have been impossible with column-extracted DNA

Lesson: For long-read sequencing and genome assembly, magnetic bead extraction is non-negotiable.

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Case Study 3: Insecticide Resistance Monitoring (Field Surveillance)

Goal: Genotype kdr mutations in Anopheles gambiae populations across 50 villages Requirements:

• PCR amplification of kdr locus (500 bp)
• Sanger sequencing or restriction digest (PCR-RFLP)
• Process 10 mosquitoes per village (500 total samples)
• Fast turnaround (results needed for spray decisions)

Method Chosen: Column extraction (96-well format) Reasoning:

• Genotyping is PCR-based (HMW DNA not needed)
• 500 samples requires high-throughput method
• Time-sensitive (results inform control strategies)
• Cost-effective (surveillance budget limited)

Workflow:

1. Extract DNA from 500 mosquitoes using 96-well column format

2. PCR amplify kdr locus in 96-well plates

3. Digest with restriction enzyme (or Sanger sequence)

4. Score genotypes (susceptible, heterozygous, resistant)

Outcome:

• Total processing time: 2 days for 500 samples
• PCR success rate: 97%
• Cost: $2,750 (500 samples × $5.50)
• Actionable results delivered to control program within 1 week

Lesson: For high-throughput, PCR-based applications, column extraction is the only practical choice.

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Summary Table: Method Selection Guide

Application	Recommended Method	Fragment Size Needed	Throughput	Time Priority	Cost Sensitivity
PCR (<5 kb)	Either (prefer columns for high-throughput)	Not critical	High	High	Moderate
PCR (>5 kb)	Magnetic beads	>20 kb	Low	Moderate	High
Sanger Sequencing	Either	Not critical	High	High	Moderate
Illumina Sequencing	Either	Not critical	High	Moderate	Moderate
PacBio Sequencing	Magnetic beads (required)	>40 kb	Low	Low	Low
Nanopore Sequencing	Magnetic beads (preferred)	>20 kb	Low	Low	Low
Genome Assembly	Magnetic beads (required for long reads)	>40 kb	Low	Low	Low
SNP Genotyping	Columns (96-well format)	Not critical	Very High	High	High
DNA Barcoding	Columns (high-throughput)	Not critical	Very High	High	High
Resistance Monitoring	Columns (high-throughput)	Not critical	Very High	High	High
Structural Variants	Magnetic beads (required)	>50 kb	Low	Low	Low
Metagenomics	Either (depends on sequencing platform)	Varies	Moderate	Moderate	Moderate

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Key Takeaways

1. Application Drives Method Choice

The most important question is: What will you do with the DNA?

• PCR-based applications: Either method works (choose based on throughput/cost)
• Long-read sequencing: Magnetic beads required (no alternative)
• Genome assembly: Magnetic beads required (HMW DNA essential)

2. Fragment Size is Critical for Some Applications

When fragment size doesn't matter:

• Standard PCR (<5 kb amplicons)
• Sanger sequencing (post-PCR cleanup)
• Illumina sequencing (fragments DNA during library prep)

When fragment size is critical:

• Long-range PCR (>5 kb amplicons) - need >20 kb template
• PacBio sequencing - need >40 kb, optimal >100 kb
• Nanopore sequencing - need >20 kb, optimal >50 kb
• Genome assembly - need >40 kb for scaffolding

3. Throughput vs. Quality Trade-Off

High-throughput applications (>50 samples):

• Columns win (96-well format enables parallel processing)
• Acceptable quality trade-off (10-50 kb sufficient for most applications)

Low-throughput applications (<20 samples):

• Magnetic beads win (better yield, purity, HMW DNA)
• Worth the extra time investment

4. Cost Considerations

Per-sample cost:

• Magnetic beads cheaper (~$3/sample)
• Columns more expensive (~$5/sample)

Equipment cost:

• Magnetic rack: ~$50-100 (affordable)
• Disruptor Genie: ~$1,000 (still expensive but half the incorrect price)

If you don't have Disruptor Genie:

• Magnetic beads more cost-effective overall
• Can use manual grinding or liquid nitrogen lysis

5. Time Investment

When time is critical (surveillance, diagnostics):

• Columns win (30 min vs. 2 hours)
• Can process 96 samples in 2 hours

When quality matters more than time (research projects):

• Magnetic beads worth the extra time
• Better purity and HMW DNA improve downstream success

6. Risk Management

If uncertain about downstream application:

• Default to magnetic beads (provides maximum flexibility)
• HMW DNA works for all applications (can be fragmented if needed)
• Column DNA cannot be "un-fragmented"

If sample is irreplaceable (museum specimen, rare species):

• Use magnetic beads (higher yield, better recovery)
• Can use portion for Illumina, save remainder for future long-read sequencing

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Practical Decision Matrix

Use this matrix to quickly decide which method to use:

 Need HMW DNA (>40 kb)?
 |
 YES ────┴──── NO
 | |
 MAGNETIC BEADS Processing >50 samples?
 |
 YES ────┴──── NO
 | |
 COLUMNS Time limited?
 |
 YES ────┴──── NO
 | |
 COLUMNS Budget limited?
 |
 YES ────┴──── NO
 | |
 MAGNETIC BEADS Either method
 (personal preference)

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Further Reading and Resources

Recommended Literature

1. Comparison of DNA extraction methods for arthropods:

- Schiebelhut, L. M. et al. (2017). "Decimation by DNA extraction: A comparison of six methods." Frontiers in Marine Science, 4, 128.

2. HMW DNA extraction for long-read sequencing:

- Mayjonade, B. et al. (2016). "Extraction of high-molecular-weight genomic DNA for long-read sequencing of single molecules." BioTechniques, 61(4), 203-205.

3. Magnetic bead technology:

- Berensmeier, S. (2006). "Magnetic particles for the separation and purification of nucleic acids." Applied Microbiology and Biotechnology, 73(3), 495-504.

4. Mosquito-specific protocols:

- Kumar, N. P. et al. (2007). "DNA barcodes can distinguish species of Indian mosquitoes." Journal of Medical Entomology, 44(1), 1-7.

Protocol Manuals

• BioDynami Genomic DNA Extraction Kit Manual
• Zymo Quick-DNA Tissue/Insect Microprep Kit Manual
• PacBio Template Preparation Guide
• Oxford Nanopore Genomic DNA Sequencing Kit Guide

Online Resources

• BOLD Systems (Barcode of Life Database): http://www.boldsystems.org

- Protocols for DNA barcoding, including extraction methods

• PacBio DevNet: https://www.pacb.com/support/documentation/

- Requirements for HMW DNA for long-read sequencing

• Nanopore Community: https://community.nanoporetech.com

- User experiences with different DNA extraction methods

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Conclusion

Choosing between column-based and magnetic bead DNA extraction is not a matter of "better" or "worse" - it is a matter of matching method to application. Both methods work well for mosquito DNA extraction, but they excel in different contexts:

Use columns when:

• Throughput is priority (>50 samples)
• Time is limited (need results quickly)
• Application is PCR or Sanger sequencing
• HMW DNA is not required

Use magnetic beads when:

• Long-read sequencing is planned
• HMW DNA is essential (>40 kb)
• Sample size is small (<20 samples)
• Maximum yield is needed from limited material
• Budget favors lower per-sample cost

When in doubt, choose magnetic beads - they provide maximum flexibility for future applications, better yield, and higher purity. The extra time investment (90 minutes) is often worthwhile insurance against needing to re-extract samples later.

Remember: DNA extraction is the foundation of all downstream molecular work. A poor extraction cannot be rescued by downstream optimizations. Choose your method carefully based on your specific application, and you will set yourself up for success.

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Document prepared for ENTM201L - General Entomology Laboratory UC Riverside, Department of Entomology Fall 2025