How To Use GCRC Genetic Resources John A Phillips III, MD Director, GCRC Genetics Vanderbilt Kelly A Taylor, MS, CGC Program in Human Genetics Cara B Sutcliffe,
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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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1965 1970 1975 1980 1985 1990 1995 2000 2005Year
Number of Entries in Online Mendelian Inheritance in Man (OMIM)
MIM1
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?
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