Transcript How To Use GCRC Genetic Resources

How To Use GCRC Genetic
Resources
Dec 13, 2002
John A Phillips III, MD
Director, GCRC Genetics Vanderbilt
Kelly A Taylor, MS, CGC
Program in Human Genetics
Cara B Sutcliffe, PhD
Director, Genotyping Core Lab
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 Project Proposal
GCRC Advisory Committee
GCRC Genetic Cores
DNA
Analysis
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
Number of Entries in Online Mendelian Inheritance in Man (OMIM)
Number of M IM entries
OMIM
13000
14,065 Dec 5, 2002
12000
11000
10000
9000
MIM12
8000
7000
MIM11
6000
MIM10
5000
MIM9
MIM8
4000
MIM7
MIM6
3000
MIM5
MIM4
2000
MIM3
MIM1 MIM2
1000
1965 1970 1975 1980 1985 1990 1995 2000 2005
Year
Obtain Genetic Information
dbSNP
OMIM
RefSeq
GenBank
UniGene
http://www.gentest.com/human_p45
0_database/srchh450.asp
http://medicine.iupui.edu/flockhart/in
dex.html
Genetic Studies of Families
Family History Is the Key to:

Identify those at
increased risk

Testing can be
offered to clarify
diagnosis

Preventative
medicine can be
available

Drug & other
therapy can be
offered
Genetic Studies of Families
Linkage
study
Case Parent Triad
TDT study
Sib pair
study
Linkage Studies
Association Studies
SNP A is NOT Associated with Disease
SNP B IS Associated with Disease
Drug Metabolizing Gene Scans
GVS CYP 2D6 Exon 6 C > T
ddA Trace
A/A
A/G
G/G
ddC Trace
C/C
C/T
T/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
Clinical Research
• Family is often
study unit
• Individual is study
unit
• Altruistic benefits
and motivations
• Personal benefits
and motivations
• Psychosocial
risks
• 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:
[email protected]
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 - nonorganic, 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 View
Note 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
[email protected]
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
TDT
• Familiy studies
(linkage, sib pair
&TDT)
Linkage
Sib pair
• Association studies
• Drug metabolizing
gene studies
ddA
Trace
A/A
A/G
G/
G
Putting It All Together (AUG-TER)
Family Chromosome
Studies
Interval
Candidate
Genes
Genes in Interval
1. ESTs, unidentified
2. ESTs, unidentified
3. ESTs, highly similar
to patched
[Drosophila
melanogaster]
4. Phosphofructokinase
(PFK)
5. BMPR2 Positional
*
& Functional
candidate for PPH
6. ESTs, unidentified
7. Deleted in pancreatic
cancer 1 (DPC1)
8. ESTs, unidentified
Genetic
Mapping
Computer
Search
Disease
Mutation
Met A A Met
TT
GG
Val G G Val
TT
CC
Ser T T Ser
CC
AA
Leu C C Leu
TT
GG
Gln C T
AA
STOP
AA
Pro C C
CC
GG
Cys T T
GG
TT
Mutation
Detection
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?