How To Use GCRC Genetic How To Use GCRC Genetic ResourcesResources

John A Phillips III, MDDirector, GCRC Genetics Vanderbilt

Kelly A Taylor, MS, CGCProgram in Human Genetics

Cara B Sutcliffe, PhDDirector, Genotyping Core Lab

Dec 13, 2002

Goals of GCRC Genetics

• Provide genetic consultations to help GCRC Investigators

• Provide comprehensive genetic lab services to GCRC Investigators

• Provide education in human genetics to GCRC Investigators & trainees

• Evaluate repository of GCRC clinical data for future genotype phenotype studies

Organization of GCRC Genetics Initiative

GCRC Advisory Committee

GCRC Project Proposal

GCRC Genetic Subcommittee

GCRC Genetic Cores

Subjects DNA Analysis

Data

Analysis

Genetic Consultation

How To Use GCRC Genetic Resources

• Genetic approach to GCRC studies

• How to get help on your genetic IRB on GCRC

• How to have DNA isolated & genotyped on GCRC

• Putting it all together (AUG-TER)

Genetic Approach to GCRC Studies

• Obtain genetic information

• Familiy studies (linkage, sib pair &TDT)

• Association studies

• Drug metabolizing gene studies

• How to get started & educational materials

Obtain Genetic Information

MIM2MIM3MIM4

MIM5MIM6

MIM7MIM8

MIM9MIM10

MIM11

MIM12

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Number of Entries in Online Mendelian Inheritance in Man (OMIM)

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OMIM

14,065 Dec 5, 2002

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Obtain Genetic Information

OMIM

RefSeq

GenBank

UniGene

dbSNP

http://www.gentest.com/human_p450_database/srchh450.asp

http://medicine.iupui.edu/flockhart/index.html

Midazolam?

Genetic Studies of Families

Family History Is the Key to:

Identify those at Identify those at increased riskincreased risk

Testing can be Testing can be offered to clarify offered to clarify diagnosis diagnosis

Preventative Preventative medicine can be medicine can be availableavailable

Drug & other Drug & other therapy can be therapy can be offeredoffered

Genetic Studies of Families

Linkagestudy

Sib pair study

Case Parent TriadTDT study

Linkage Studies

Association Studies

SNP A is NOT Associated with Disease

SNP B IS Associated with Disease

Drug Metabolizing Gene Scans

ddA Trace

ddC Trace

A/AA/GG/G

C/CC/TT/T

GVS CYP 2D6 Exon 6 C > T DNA Sequencing CYP2D6 Exon 6 C > T

How to Get Started

• Obtain genetic information about your GCRC study from John Phillips

• Consider which genetic approach to use (linkage, sib pair, TDT or association) & discuss with John

• Consider which drug metabolizing genes are important

How to Get Started

• Submit IRB & GCRC study but ask for DNA collection

• Have GCRC isolate & store DNA for you

• Discuss genotyping with John & submit GCRC Genotyping Application

How to Get Started • GCRC Genotyping Application Form:

– study design– genotype(s) requested– Rationale for genotyping – Number of genotypes – Overlap with other support– Timeframe

• Educational materials: www.mc.vanderbilt.edu/GCRC

Genotyping Application Forms

How To Use GCRC Genetic Resources

• Genetic approach to GCRC studies

• How to get help on your genetic IRB on GCRC

• How to have DNA isolated & genotyped on GCRC

• Putting it all together (AUG-TER)

GCRC Genetics Ascertainment Services

Kelly A Taylor, MS, CGC

Program in Human Genetics

Vanderbilt University Medical Center

Differences Between Genetic Research and Clinical Research

Genetic Research

• Family is often study unit

• Altruistic benefits and motivations

• Psychosocial risks

Clinical Research

• Individual is study unit

• Personal benefits and motivations

• Medical risks

Differences Between Genetic Research and Clinical Genetic Services

Research• Identify mutation• No charge• No results

disclosure• No direct benefit• Education and

referral

Clinical• Use mutations for

diagnosis• Fee for service• Interpretation of

results• Clinically relevant

information• Genetic counseling

What Unique Issues Need to Be Considered

in Developing an Ascertainment Protocol

for a Genetic Study?

Issues To Be Addressed During the Informed Consent Process

� Risks � DNA Ownership and Banking� Secondary Usage of DNA samples� Disclosure of Results� Incidental Findings� Confidentiality� Recontacting Participants� Duty to Warn

How Do I Add A Genetic Component to My Study?

• Get IRB approval

• Enroll patient

• Informed consent

• Blood sample

• Collect additional information

• Family history

How Can Kelly Help?

• IRB development

• Provide templates, answer questions

• Write IRB proposals

• Ascertainment and enrollment of participants

• Training of staff

• Blood collection kits

How Do I Contact Kelly?

Kelly A Taylor, MS, CGC

Phone: 322-7195

Email: ktaylor@phg.mc.vanderbilt.edu

Website: http://phg.mc.vanderbilt.edu/fac

How To Use GCRC Genetic Resources

• Genetic approach to GCRC studies

• How to get help on your genetic IRB on GCRC

• How to have DNA isolated & genotyped on GCRC

• Putting it all together (AUG-TER)

DNA Isolation and Genotyping

Cara Sutcliffe

DNA Resources Core

Vanderbilt University

An Overview

•Samples and Data Collected

•Experimental Coding Assigned

•Samples Processed

•Samples and Data Used in Project

Principles for Process Development• Information Integrity

– Confidentiality– Accuracy

• Sample Integrity– High yield of Appropriate Quality DNA– Controlled, Rapid, and Accurate Retrieval

• Practical Issues– Safety– Cost– Flexibility

Step One - Sample Arrives

• Sample Arrival - through delivery company or with a submitter from clinic or the field

• Record Keeping - information supplied by the submitter as well as qualitative determinations made by the core are recorded.

• Initial Sample Manipulations - print labels, initiate tracking forms, freeze aliquots, spot blood cards, complete log information

Step Two - DNA Extraction• Possible Methods

– Organic - “Classic” phenol/chloroform, simple but involves hazardous chemicals

– Binding Column - Clean DNA, but not scalable– Silica Resin - scalable and inexpensive, but DNA is generally lower

molecular weight– Lytic Preps - fast, inexpensive, and simple, but DNA is impure and thus

subject to degradation– Salting Out – Magnetic Binding

Puregene• The Puregene™ extraction kit by Gentra

Systems is a modified salting out procedure - non-organic, scaleable, and extensively tested.

• In addition to being scaleable, the protocol easily lends itself to both automation and semi-automation for higher throughput and cost savings.

• DNA Stability data and additional protocols are available via PDF from the Gentra website.

MagnaPure– Surveyed to determine demand and price

point for small (<1ml) volume extractions– System is capable of extracting DNA OR

RNA from 32 samples in ~2 hours – In addition, the Magnapure sets up Roche

Lightcycler reactions for real time PCR/SNP assays and can be programmed to set up 96 well plates for other genotyping assays.

MagnaPure II• Nucleic acids can be isolated

from whole blood, cultured cells, buccal cells.

• Samples are lysed, magnetic glass particles are added and the nucleic acids bind to them. Cellular debris is removed during washing steps. The purified DNA is eluted.

• Post-elution protocols provide for dilutions or assay set up.

Step Three - Quantitation• Samples are quantitated using

fluorometry.

• Fluorometry is initially performed using a Hoescht dye assay for dsDNA on the Hoefer DyNAQuant 200™. Sample readings <30ng/ul are confirmed by quantitative PCR using the RNaseP primer/probe set

Step Four - Storage• Sample storage is secured.

• -80° and Liquid Nitrogen freezers are monitored by dial out alarm system for malfunctions.

• Orderly and intuitive racking.

– VSN also serves as locator for DNA vials

– Other samples are banked chronologically and tracked electronically with hardcopy backup.

Step Five - Requisition• Must be authorized by the investigator

using the Core’s database. The database records each requisition, tracking remaining amounts and recipients.

• VSN and Genetic Ids are matched to confirm delivery of the correct sample

• Color-coded storage vials aid in accurate retrieval

Quality Control• DNA

– Observed and Expected Yields are calculated and reviewed on every sample

– Agarose gels are run on a subset of samples to confirm concentration and molecular weight

– Bar Coded Labels are used on forms & sample containers

– Color coded DNA storage vials • Data

– 2X Proofreading– Daily Backup– Multiple levels of Password Protection

A Laboratory ViewNote the

• Presence and Use of Safety

Devices– Face and Splash Shields– Nitrile gloves– Spill Tray– “One Hand” Tube Rack

• Color and Bar Coded Storage Tubes

• Bar Coded/Labeled Processing tubes

Genotyping Using the 7900HT• The ABI PRISM® 7900HT: real-time PCR system that detects & quantitates nucleic

acid sequences. Automation & 384-well plate capability allow for very high-throughput. • Interchangeable formats (96 & 384 well) provide flexibility• Hand-held and integrated bar code readers simplify sample tracking • Wavelength detection from 500-660nm allows the use of multiple fluorophores in a

single reaction • Candidate gene/region association and linkage mapping studies are approachable with

this technology

Assays-On-Demand•Assays-on-Demand are approximately 200,000 “on the shelf” human SNP assays•Designed around high allelle frequency SNPs identified in multiple databases, the assays use fluorogenic 5' nuclease chemistry and TaqMan probes. •Validated on 90 individual DNAs to ensure biological relevance

•Allele frequencies are available for 4 ethnic groups•Convenient online batch searching and ordering •A sophisticated bioinformatics pipeline selects SNPs and designs assays

Assays-by-Design• Assays-by-Design is a custom design service providing validated

assay products for gene expression and SNP genotyping. You submit your target sequence, and ABI returns a QC-verified, all-in-one tube assay ready to use with TaqMan® Universal PCR Master Mix.

• Using Assays-by-Design eliminates the manual and technology-specific task of designing primers and probes

• Cost per data point comparable to "build your own" • One-tube assays remove the steps of balancing primers and

probes and optimizing reactions

Core Statistics

• Over 29,000 samples• 75 Projects Ongoing for 40

Investigators• Approximately 30 Services Offered • 6 Multi-Center Projects• ~130 liters of blood processed

For Further Information• For A More Complete List of Services and Pricing

See our Website (http://phg.mc.vanderbilt.edu) DNA Resources Core Section

• For Project Planning contact Cara Sutcliffe- 936-2744 or cara@phg.mc.vanderbilt.edu

Thanks to the DNA Resources Core students and staff: Maria Comer, Derek Gatta, Lindsey Herrel, Elizabeth Matthews, Allison Mills and Kate Redding

How To Use GCRC Genetic Resources

• Genetic approach to GCRC studies

• How to get help on your genetic IRB on GCRC

• How to have DNA isolated & genotyped on GCRC

• Putting it all together (AUG-TER)

Putting It All Together (AUG-TER)

• Where to get application forms

• Types & size of studies that fit GCRC

• Who to contact:

Genetic info & approaches: John Phillips

IRBs & consents: Kelly Taylor

DNA isolation: Nina Smith & Cara Sutcliffe

Genotyping - Cara Sutcliffe

Genotyping Application Forms

Types & Size of GCRC Studies

• Familiy studies (linkage, sib pair &TDT)

• Association studies

• Drug metabolizing gene studies

Linkage

Sib pair

TDT

ddA Trace A/A

A/GG/G

Genetic Mapping

FamilyStudies

Chromosome Interval

Met A A Met T T G GVal G G Val T T C C Ser T T Ser C C A ALeu C C Leu T T G G Gln C T A A A APro C C C C G GCys T T G G T T

STOP

*

1. ESTs, unidentified

2. ESTs, unidentified

3. ESTs, highly similar to patched [Drosophila melanogaster]

4. Phosphofructokinase (PFK)

5. BMPR2 Positional BMPR2 Positional & Functional & Functional candidate for PPHcandidate for PPH

6. ESTs, unidentified

7. Deleted in pancreatic cancer 1 (DPC1)

8. ESTs, unidentified

ComputerSearch

MutationDetection

Candidate Genes

DiseaseMutation

Putting It All Together (AUG-TER)

Genes in Interval

GCRC Genetic Resources• GCRC Genetics Website:

www.mc.vanderbilt.edu/GCRC

• Who to contact for help:

Genetic info & approaches: John Phillips

IRBs & consents: Kelly Taylor

DNA isolation: Nina Smith or Cara Sutcliffe

Genotyping - Cara Sutcliffe

GCRC Genetic Studies Can Help

Questions About GCRC Genetics?