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| High Throughput Fragment Analysis |
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Understanding genetic variations and their relationship to biological function is a key area of today's genomic research. As prolific amount of sequencing data gets generated every day, more and more genetic marker phenotype relationships which require further investigations become available. Large scale genomic scans using microsatellite and SNP markers and studies involving mutation scans for a variety of biological scenario require high throughput and quality controlled fragment analysis techniques. The Automated Fragment Analysis Services in TCGA has been developed with the objective to provide high throughput data of assured quality to researchers working in such projects. The services offered range from whole genome scans using microsatellite markers to custom marker scans as well as SNP and mutation scans using SNaPshot technology. Data generation is based on fluorescent detection of fragments in 3730 Genetic Analyzers and subsequent automated analysis using GeneMapper. Any data of suboptimal quality is detected and flagged for confirmation. The services are highly adaptable and have versatile options to suit every project requirement. End to end services are available including assay design and optimization as well as DNA and RNA extraction from samples to complete data analysis up to allele calls. Technical Support is available round the clock to understand requirements of investigators and provide inputs to customize services required.
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| Services Available |
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Microsatellite Genotyping
Complete human genome scan based on 811 microsatellite markers (10 cM and 5 cM
sets) as well as mouse genome scans based on Linkage kits from Applied
Biosystems are available. We also provide genotyping services for Genethon 1 cM
markers for human genome. The assays are optimized for high quality and
automated genotyping and are available in a ready to go format consisting of 17
plex runs.
Custom Microsatellite Genotyping
We provide microsatellite genotyping for custom microsatellite markers for any
genome. The end to end service consists of fluorescent Oligonucleotide
synthesis, multiplex assay design, optimization and sample processing, based on
5 dye detection and subsequent data analysis.
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Mutation Scan
High throughput multiplex mutation scans are available based on SNaPshot
technology. We offer SNP validation, mutation scans and Restriction Fragment
Analysis in this module.
Custom Fragment Analysis
AFLP, tRFLP, PCR based analysis and any other custom fragment analysis by
electrophoresis and fluorescent detection available. We provide round the clock
Technical Support for planning and customizing any application.
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| Technology |
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3730 Applied Biosystems
Genetic Analyzer
We have two state of the art
3730 Genetic Analyzers from
Applied Biosystems which can
generate 2 mb sequencing data
per day.
Hydra, Biomek &
Multimek Robotic Workstations.
We have multiple robotic
workstations to scale up sample
processing and reaction set up
to high throughput levels. We
have Hydra (Robins), Biomek and
Multimek (Beckman) for robotic
transfers and set ups.
9700 PCR Thermal Cycler
We use multiple 96-well and dual
384-well 9700 PCR Thermal
Cyclers, the industry standard
tool for high throughput PCR.
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| FAQs |
1. How much DNA do I submit? At what concentration?
We ask you to submit 50 to 100 ng of genomic DNA per PCR.
2. How should I submit DNA for PCR?
8 or fewer samples: Please submit them in 0.2-0.5-mL micro centrifuge tubes sealed with parafilm.
16 or more samples: You may submit them in 8 tube strip tubes or 96-well plates.
All strip caps and plate covers must be securely fastened to prevent evaporation and/or leakage from the tubes.
3. How should I label my tubes?
Please label tubes containing DNA samples with the name of the sample. Likewise, label tubes containing primers with the name of the primer. Importantly, fill in the sample names and primer concentrations in the sample submission form.
4. What is the best way to determine the concentration of my DNA sample?
Verify the concentration of your sample on an agarose gel along with a size standard ladder to get a fairly good idea of the concentration and size of your DNA. We perform Pico Green quantitation of DNA before using the samples for PCR.
5. How should I prepare and purify my DNA for PCR?
By any standard method that generates DNA of sufficient quality for PCR.
6. What is the size range of fragments that can be detected in Fragment Analysis?
50 to 500 bp. If you expect fragment of sizes outside this range, please contact techsupport@tcgaresearch.org.
7. How much multiplexing can I do?
We use 5 dye detection. So, we can multiplex 4 colors at a a time (fifth color is for size standard). Furhter, we can multiplex your fragments by size if possible. Overall, we detect 17-plex microsatellites and 6 to 10 plex SNaPShot products routinely.
8. What is the accuracy of sizing in Fragment Analysis and how is it different from my manual gels?
The accuracy of sizing provided in our analysis is +/- 0.5 bp. We can achieve such high levels of accuracy and automation in sizing because we include size standard in every sample, unlike manual gels where size standard is loaded in in only one lane of the gel.
9. Can TCGA supply flurescent labeled primers for PCR?
We can synthesize fluorescent labeled primers for PCR and Fragment Analysis.
10. How can I obtain TCGA Fragment Analysis order form?
The order form is available on our website www.tcgaresearch.org or call and we will e-mail or fax it to you.
11. Do I have to fill out the whole sample sheet?
Yes, you need to fill the submission form.
12. How will I get Fragment Analysis data?
We provide both excel files of sizes and alleles as well as PDF files of traces for viewing and printitng purposes.
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Related links |
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www.appliedbiosystems.com
(for 3730 Genetic Analyzer)
www.geospiza.com (for downloading sequence visualization software Finch TV)
www.adobe.com/products/acrobat
(for acrobat reader)
www.ncbi.nlm.nih.gov/
(for Blast search)
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