Molecular Core FacilityMolecular Core FacilityMolecular Core Facility

Grice Marine LaboratoryCollege of Charleston

OverviewOverviewOverviewThe Molecular Core Facility was established in the spring of 2002 and formally opened in March 2003. The facility is available to CofC faculty, students, interns and visiting scientists. DNA sequencing services are also offered to scientists throughout the Fort Johnson research complex and other local institutions on a fee per service basis. Funding for the facility was provided through a grant from the NSF. The CofC Biology Department and GML have also provided support for the facility.

Available EquipmentAvailable EquipmentAvailable Equipment• Beckman CEQ 8000

automated DNA sequencer

• Versa Doc imaging system

• Thermal cycler• Microcentrifuge• Electrophoresis

workstation

• PCR workstation • Gel dryer• Vacuum

concentrator• Fluorometer• Refrigerated

centrifuge• Water bath, heat

block, vortex, rocker

Beckman CEQ 8000Beckman CEQ 8000

ServicesServicesServices

• Cycle sequencing reactions

• Capillary electrophoresis – Beckman CEQ 8000

• Fragment analysis– AFLPs– microsatellites

Fee TableFee TableFee Table

Electrophoresis Electrophoresis OnlyOnly

Cycle Cycle Sequencing and Sequencing and ElectrophoresisElectrophoresis

ReRe--Run Failed Run Failed SequenceSequence

AFLP AFLP SequencingSequencing

InternalInternal(CofC Biology Department)

$2.50 $8.00 $6.00 $3.50

IntermediateIntermediate(Non-Biology CofC

faculty and Ft. Johnson scientists)

$4.00 $9.00 $6.00 $6.00

ExternalExternal $7.00 $11.00 $6.00 $8.00

How is DNA prepared for sequencing?How is DNA prepared for sequencing?

1. Extract DNA from organism2. Amplify region of interest by PCR3. Purify DNA4. Quantify DNA 5. Cycle sequence DNA6. Capillary electrophoresis

Why is it important to have pure DNA?Why is it important to have pure DNA?

• Impure template preparations can inhibit the cycle sequencing reaction.

• Any negatively charged impurities may also be co-injected with your sample into the capillary.

• These undesirable particles will result in low signal and erratic or crashed current profiles.

Purification MethodsPurification Methods

• Commercial kits– Qiagen DNA isolation kits– Promega Wizard

• USB ExoSAP IT™• Agarase digestion

– This method can be used when multiple bands are seen on the gel.

Other commercial kits and purification methods may be used; however, residual alcohol, salt and phenol will interfere with the sequencing reaction. CsCl Preps have been found to cause low signal and current instability.

Why is it important to quantify DNA?Why is it important to quantify DNA?

• Adding the appropriate amount of DNA is essential for an efficient cycle sequencing reaction. – The template to primer ratio is also important and should be

at least 40:1.

• Too much template can clog the capillary causing an erratic or crashed current. It may also yield a very high signal and inaccurate base calls.

• Too little template will yield a very low signal and inaccurate base calls.

Quantification MethodsQuantification Methods

• It is strongly recommended that samples be quantified on an agrose gel using a mass ladder.

• If a spectrophotometer or flourometer will be used to quantify DNA the sample should be read and indicate the same concentration at least three times. A known standard should also be used to calibrate the machine before and after all samples are read.

Amount of DNA Required for Cycle Amount of DNA Required for Cycle Sequencing ReactionsSequencing Reactions

dsDNA 50 - 100fmolssDNA 25 - 50fmolPurified PCR product 10 - 50fmol

Size(kilobase pairs)

ng for 25 fmol ng for 50 fmol ng for 100 fmol

0.2 3.3 6.5 130.3 4.9 9.8 200.4 6.5 13 260.5 8.1 16 331.0 16 33 652.0 33 65 1303.0 50 100 1954.0 65 130 2605.0 80 165 3256.0 100 195 3908.0 130 260 520

10.0 165 325 65012.0 195 390 78014.0 230 455 91016.0 260 520 104018.0 295 585 117020.0 325 650 130048.5 790 1575 3150

For ssDNA, the values (ng) should be divided by 2.

Primer ConsiderationsPrimer Considerations

• Template to Primer ratio should be at least 40:1• Size should be around 20 bases • The optimal Tm is 60ºC but it should be at least

50ºC• GC content should be approximately 50%

– Primers with a high annealing temperature, such as those with GC clamps, may be used in a two step cycle sequencing reaction, which combines annealing and extension steps.

• At least 2µL of a 5 – 10µM solution should be provided for each reaction

What is cycle sequencing?What is cycle sequencing?What is cycle sequencing?

An amplification reaction that is carried out in a thermal cycler in which only one primer is

added to the reaction. The product is single stranded DNA of which the terminal

nucleotide is labeled with a fluorescent dye.

Cycle Sequencing Reaction Cycle Sequencing Reaction ComponentsComponents

CEQTMDTCS Quick Start Mix includes all of the reagents necessary for cycle sequencing

except the template DNA, primer, and dH2O.

• Nucleotides (dATP, dCTP, dTTP, dITP)• Fluorescently labeled di-deoxy nucleotides (ddUTP,

ddGTP, ddCTP, ddATP)• Tris –HCl, MgCl2, reaction buffer (pH 8.9)• DNA polymerase (Thermo Sequenace)• Pyrophosphatase

Cycle Sequencing ReactionCycle Sequencing Reaction

Original template sequence TGGCATATGGCATA

Primer AnnealingPrimer Annealing

Nucleotide ExtensionNucleotide Extension

Termination of DNA ReplicationTermination of DNA Replication

Post Reaction CleanPost Reaction Clean--upupEthanol Precipitation

• A stop solution is added to the reactions to terminate DNA amplification.– Components: 1.5 M NaOAc, 50 mM EDTA, 20mg/mL

glycogen

• The DNA is precipitated by adding 95% ethanol to the sample and centrifuging at 14,000 rpm at 4ºC for 15 minutes.

• The DNA pellet is rinsed two times with 70% ethanol and dried in a vacuum. It is then re-suspended in de-ionized formamide.

How is a sequence generated by How is a sequence generated by capillary electrophoresis?capillary electrophoresis?

Migration Time

Migration Rate Towards Detector

AC* ACC*

T1 T2 T3

Laser

Anode +

Sequence generated Sequence generated AACCCCGG

A*

ACCG*

T4

ResultsResults

Results can be provided in the following formats

Results can be provided in Results can be provided in the following formatsthe following formats

1. CEQ 8000 database (.ceq) - Files can only be opened in Beckman CEQ analysis software.

2. Standard Chromatogram Format (.scf) -Files can be opened in most sequence analysis software programs (e.x. BioEdit, TraceViewer, Chromas).

3. Text only (.txt)4. Sequence results (.seq) 5. FASTA (.fasta) 6. PHRED (.phd.1) - Produces a PHRED

and a .scf file7. Electropherogram raw data only (.esd)

Troubleshooting ToolsTroubleshooting Tools

Profile starts high, gradually decreases insignal strength as the run progresses

Profile starts high, gradually decreases insignal strength as the run progresses

Current ramps up to thefinal separation level inthree stages.

Current remains steadythroughout the rest of theseparation time

Current ramps up to thefinal separation level inthree stages.

Current remains steadythroughout the rest of theseparation time

Raw Data AnalysisRaw Data Analysis

Good signal Intensity = 25,000 – 130,000cnts

• Low signal intensity <10,000cnts

• Very low signal intensity (poor quality) <5,000cnts

• High signal intensity >130,000cnts

Low SignalLow Signal

Type 1 – Normal current, unincorporated dyes seen

Type 2 – Normal current, no unincorporated dyes seen

• Not enough template

• Poorly designed primer

• Not enough primer

• Reaction Inhibitor

• Expired reagents

• Bad formamide

High SignalHigh Signal

• Too much template

Excess SaltExcess Salt

Mixed Template or Mixed Template or Multiple Sequence EventsMultiple Sequence Events

Current AnalysisCurrent Analysis

• Should ramp up in three stages and remain steady.• Injection inhibitor is impeding the flow of current

through the capillary.– Salt, protein, ethanol, too much DNA, large or supercoiled

DNA

Current AnalysisCurrent Analysis

• Gradually declining current– Supercoiled DNA, no

pre-heat treatment

Current AnalysisCurrent Analysis

• Erratic current– Bubble in capillary or manifold

Guidelines for TroubleshootingGuidelines for Troubleshooting

• Low signal + normal current = Too little DNA, poor primer design

• Low signal + current crashes = Salt, protein, ethanol, large or supercoiled DNA

• Low signal with no unincorporated dyes + normal current = Expired or bad reagents

• High signal + current crashes = Too much DNA• Peaks within peaks in analyzed data = Overranged raw

data or multiple sequence events• Red and Black dye drop out + normal current +

Low signal = Bad formamide• Erratic current + no signal = Bubble in manifold

Fragment AnalysisFragment AnalysisFragment Analysis

DNA Fragment Analysis is simply DNA sizing.

Fragment Analysis includes detection of AFLPs, Microsatellites and SNPs.

Sequencing Sequencing vsvs Fragment AnalysisFragment Analysis

Sequencing• Linear amplification

– No new template generated

• One unlabeled primer• Templates are generally

plasmids and PCR products

• Sequencing polymerase• Dye labeled terminators

Fragment Analysis• Exponential

amplification– New template generated

each cycle• One unlabeled and one

labeled primer• Template is genomic

DNA• PCR polymerase• No dye labeled

terminators– Dye labeled primer

Fragment Analysis DataFragment Analysis Data

Molecular Core FacilityMolecular Core FacilityMolecular Core Facility

Grice Marine LaboratoryCollege of Charleston

Tricia Roth

MCF Lab Manager

rotht@cofc.edu

843-953-9193