Transgenomic WAVE:

The Genomics Core Facility offers three technologies for the discovery and analysis of genetic variation, the Transgenomic WAVE, the ABI 3100 Genetic Analyzer, and the Pyrosequencer. The Transgenomic WAVE is best utilized for the screening of a population for the detection/discovery of mutations. Once a mutant sample is identified, the exact mutation can be determined by sequencing the sample using the ABI 3100. And finally, the frequency of a specific mutation or SNP in a population of samples can be determined using the Pyrosequencer. Each of the technologies are describe below along with directions on how to get started and a fee structure.

Mutation Detection using the WAVE
Mutation detection is performed in the Genomics Core Facility using the Transgenomic WAVE system. The Transgenomic WAVE system utilizes DHPLC (denaturing high performance liquid chromatography) in conjunction with its patented DNASep cartridge to screen PCR amplified samples for mutations in a cost effective manner.
Contact Dr.Michael Crowley(mcrowley@uab.edu) in the Core Facility to discuss your project. Dr.Crowley has extensive experience in mutation detection using the WAVE. Speaking with him in advance will save you a lot of time and headaches.

Getting started with the WAVE

1. Amplicon and primer design
Core user:

• Design primers to amplify gene of interest
  - products 200 - 400bp (to 500bp where necessary)
  - design no closer than 50bp from sequence to be analyzed
  - DO NOT ORDER YET
  
• Compile the following information as a Word document and submit it to the Core Facility for melting curve analysis/injection temperature estimation
  - Gene sequence with your region of interest italicized
  - Highlight or underline the suggested primers
  - Indicate the total size of amplicon, the distance from 5' end of forward primer to 5' end of region of interest, and the distance from 3' end of region of interest to 5' end of reverse primer

2. Melting Curve Analysis for Injection Temperature Estimation
Core Facility:

• Input data into Wavemaker™
  o Analysis of product melting profiles
  o Formulation of appropriate elution buffer gradient (size and sequence dependent)
  o Estimation of injection temperatures (sequence dependent)
• Core will give the User the 'OK' to order primers or notify User of the need for primer modification if necessary

3. Injection Temperature Confirmation using 'Normal' Control Samples
Core User:

- Order primers on 'OK' by Core Facility
- Amplify 3 - 5 normal controls for each fragment to be analyzed
- Amplify known mutation controls where possible
- Run 5µL of each product on an agarose gel to confirm PCR amplification
- Sequence at least one PCR product of each fragment to confirm sequence
- Take 'normal control' samples to Core Facility

Core Facility:

- Inject PCR fragments at 50ºC (non-denatured) to confirm product specificity
- Inject fragments at 4 other temperatures on and above temperatures estimated from melting curve analysis
o Identifies problems with PCR reaction
o Confirms estimated DHPLC injection temperatures
o Provides chromatogram information for products at injection temperatures
o Identifies common polymorphisms

4. Submit Test Samples - DHPLC Mutation Screening
Core User:

-Amplify test samples on 'OK' by Core Facility
o minimum of 3 samples per PCR fragment (chromatogram pattern comparison)
o Always include a 'Normal' (WT) Control and a 'No Template' Control
o Include known mutation controls where available

- Provide gel photograph of PCR products and Excel file with sample details when submitting samples

Core Facility:

-Core Facility will analyze submitted samples and provide DHPLC results for requested fragments

WAVE Fees

• Project start-up costs

  o DHPLC Column $1500 (highly recommended for large projects)
  o Melting Curve Analysis - $2 per fragment (flat rate)
  o Injection Temperature Confirmation - $4 per fragment

• On-going project costs

  o 80 cents per injection

Project costs: a simplified example

• start-up
  o 20 PCR fragments for analysis in 3 normal controls
  o [20 X $2] + [3 X (20 X $4)] = $ 280
• on-going
  o 20 PCR fragments/sample with 40 injection temperatures
  (i.e. 2 injection temperatures per PCR Fragment)
  o 40 injections x $0.80/injection = $32 per sample
  

Cost Analyses for Mutation Screening Projects

DHPLC vs. Direct Sequencing - small project

11 samples x 7 PCR fragments/sample = 77 PCR products
12 injection temperatures per sample
12 temperatures X 11 samples = 132 injections total
8 PCR products with variations - 7 for sequencing (1 pair with same pattern)

DHPLC                                                                           Direct Sequencing

Start-up cost (7 PCR fragments)    = $98                   7 fragments x 11 samples (F/R) = $1540
Run Cost (132 injections)                = $105.60
Sequencing 7 PCR products(F/R) = $140       
                                                           = $343.60

DHPLC vs. Direct Sequencing - large project (67 exons)

10 samples x 80 PCR fragments(67 exons) = 800 PCR Reactions
132 injection temperatures per sample
132 temperatures X 10 samples = 1320 injections total

DHPLC                                                                          Direct Sequencing

Start-Up Cost (80 PCR fragments)     = $1120         80 frag X10 samples = $16 000
Run Cost (1320 injections)                   = $1056         (800 products, F/R)
Seq: 82 variant PCR fragments (F/R) = $1640
                                                                 = $3816

Recommended Procedures
Before you begin your PCR reactions for DHPLC, please read the following recommendations which will optimize the results from the WAVE™.

DNA Quality

• DNA must be clean!!
• Salting Out method preferred
• Ethanol Precipitation and wash recommended for spin column and chaotropic salt (guanidinium isothiocyanate) extraction methods
• Chloroform/isoamyl back extraction followed by ethanol precipitation and wash recommended for organic extraction (phenol chloroform)
• Aim: to remove all cellular debris and organic compounds

PCR

Recommended PCR protocols

Recommended touchdown PCR protocol - Minimizes formation of non-specific PCR products
1. 950C' 5'
2. 950C'30"
3. 650C'560C-30"
4. 720C' 30"
5. repeat 2-4, decreasing 10C/cycle for total of 10 cycles
6. 950C'30"
7. 530C'30"
8. 720C' 30"
9. repeat 6-8, for total of 25 cycles
10. 720C' 7'
11. 40C = infinity

Heteroduplex Augmentation Cycle - Denature and slow cool
1. 95ºC for 5 minutes
2. 93.5ºC ' 1 minute
3. repeat 2, decreasing 1.50C/cycle for total of 47 cycles
4. Cool to 4ºC

Recommended PCR reaction components

• Polymerase
  o Hotstart Taq (HotMaster Taq,Eppendorf; Amplitaq Gold, Perkin Elmer)
  o Proofreading Taq (Optimase, Transgenomic)
  " Primers
  o Final Concentration 0.2 - 1.0mM
• DNA

  Template Recommended quantity in PCR
  Human genomic DNA 50 - 200 ng
  Phage DNA 1 - 10 pg
  Plasmid DNA 0.1 - 1.0 pg

Acceptable PCR reaction additives

• Acceptable additives (maximum final concentration)
  o 10% DMSO
  o 2% Glycerol
  o 1.25-2.5M Betaine
• Additives where final concentration must be <1%
  o High molecular weight stabilizers (e.g. polyethylene glycol)
  o Detergents including
  • TritonX10
  • NP40
  • Tween 20
  • SDS/SLS

Unacceptable PCR reaction additives
The following additives will cause irreversible DNASep Cartridge Damage

• " Unidentified ingredients described as "proprietary", "stabilizers", "enhancers" or "additives"
• " Template DNA extracted/purified other than by recommended procedures
• " Mineral Oil
• " Formamide
• " Autoclaved Water
• " Proteinase K
• " Bovine Serum Albumin (BSA)
• " Loading dyes (cresol red)
  

Pyrosequencing:

SNP analysis using the Pyrosequencer
High-throughput SNP analysis using the Pyrosequencer is performed by the Genomics Core Facility in collaboration with the GCRC. The Pyrosequencing technique of SNP detection employs sequencing by DNA synthesis and detects nucleotide incorporation through a luciferase reaction. The Pyrosequencer is capable of processing a 96-well plate of samples in less than 15 minutes.

Principles of Pyrosequencing

The Pyrosequencing technique of SNP detection employs sequencing by DNA synthesis and detects nucleotide incorporation through a luciferase reaction.

1. The unique sequencing primer is annealed to the biotinlyated single-stranded DNA template and incubated with DNA polymerase, ATP sulfurylase, luciferase and apyrase and the substrates, adenosine 5' phosphosulfate (APS) and luciferin.
2. dNTPs are sequentially added to the reaction mixture in a predetermined order according to the template sequence. When DNA polymerase incorporates a nucleotide, PPi is released in an amount equimolar to the quantity of nucleotide incorporated.
3. ATP sulfurylase quantitatively converts PPi to ATP in the presence of adenosine 5' phosphosulfate. The production of ATP drives the luciferase-mediated conversion of luciferin to oxyluciferin thus producing light in quantities proportional to the amount of ATP generated. The amount of light detected is reflected as a peak in the pyrogram and is proportional to the number of nucleotides incorporated.
4. Unincorporated dNTPs and ATP are continually degraded by the inclusion of apyrase in the reaction mixture. When degradation is complete, the next dNTP in the sequence is added.
5. Subsequent dNTP additions occurs one at a time until the template to be sequenced is complete (generally 10 - 20 nt).
6. Results consist of a pyrogram in which each peak corresponds to the incorporation of a particular nucleotide, indicated above the peak, and peak height correlates with the number of nucleotides incorporated.

Outline of Pyrosequencing procedure

The sequence of your gene of interest and the position and exact identity of your SNP/mutation must be known.

1. Using primer design software, design a primer pair to amplify a small region (100 - 200bp) around your mutation.
2. Send primer and amplicon sequences to the core facility. Highlight the position and nature of SNP (i.e. missense A to C to be denoted by A/C in amplicon sequence).
3. Core Facility will use the Pyrosequencing software to design a primer to sequence across the SNP location. The Core Facility may require the amplicon primers to be altered to optimize the pyrosequencing reaction.
4. The Core Facility will confirm primer sequences and notify which primer requires biotinylation.
5. PCR your amplicon using the recommended PCR profile and reagents.
6. Electrophorese PCR products. Products must be clean (i.e. no non specific bands)
7. Provide gel photograph of PCR products and Excel file with sample details when submitting samples
8. Core facility will isolate the biotinylated strand of the PCR product and set up Pyrosequencing reactions using the biotinylated ss-DNA and SNP specific sequencing primer. Run reactions using the SNP specific sequencing profile.
9. Drink 1 cup of coffee.
10. Analyze results.

Getting started
Contact Dr.Michael Crowley(mcrowley@uab.edu ) in the Core Facility to discuss your project. Speaking with him in advance will save you a lot of time and headaches.

Reaction optimization
The following documents provide excellent advice on primer design and PCR optimization specifically for Pyrosequencing. It is highly recommended that Pyrosequencer users read these documents.

PCR optimization for Pyrosequencing ( PDF )

Primer design for Pyrosequencing ( PDF )

Fees
To be determined