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Breast Cancer Screening Strategy & the Transfer of New Diseases to Automated PCR & Sequencing

Breast Cancer Screening Strategy & the Transfer of New Diseases to Automated PCR & Sequencing. Natalie Morrell, MTO3 Birmingham. Summary. Previous BRCA screening techniques Why strategy had to change New BRCA Strategy Results so far Transfer of new Diseases Overall Impact of Automation.

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Breast Cancer Screening Strategy & the Transfer of New Diseases to Automated PCR & Sequencing

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  1. Breast Cancer Screening Strategy & the Transfer of New Diseases to Automated PCR & Sequencing Natalie Morrell, MTO3 Birmingham

  2. Summary • Previous BRCA screening techniques • Why strategy had to change • New BRCA Strategy • Results so far • Transfer of new Diseases • Overall Impact of Automation

  3. dHPLC Sequencing PTT SSCP Breast Cancer Before Automation……

  4. Reasons for Change • Genetics White Paper Guidelines, 2004 • By June 2006: • All new referrals for large gene screening must be reported within 8 weeks • BRCA, HNPCC, FAP, Gorlins etc

  5. New Breast Cancer Screening Strategy… • Sequencing of BRCA1 & BRCA2 genes • BRCA 1 has 24 exons (22 coding) • BRCA 2 has 27 exons (26 coding) • Total of 48 coding exons • 16,190 bases • MLPA

  6. PrimerOptimisation • All primers work at concentration of 12.5pmol/µl • Anneal at 58ºC • 30 cycles 63 Fragments ranging in size from 243bp to 896bp

  7. Breast Cancer Panels • Each panel consists of 3 patients • 63 fragments and a negative control split over two 96 well plates.

  8. Breast Cancer Plate Format

  9. Biomek NX robots Pre PCR work Post PCR work Biomek FX robot 2 ABi 3730 Sequencers Introduction of New Equipment

  10. Manual dilution of primers into 0.5ml tubes Robot aliquots 1µl of each primer into appropriate wells on plates (inc Negative) Primer spotted plates put in drying oven for 30 mins Plates sealed and frozen until required Batch test performed before plates are used for patient samples 2 Normal controls and 1 Negative control used to check for contamination and also that primers amplify ok Run Check gel (all wells for 1 Normal control & Negative Control) Primer Spotting & Batch Tests

  11. Report issued DNA samples activated If needed Plate-based Sequencing MLPA PCR using primer-spotted plates Gap fill PCRs Pre-PCR NX Log sheet entry Mini gel check On-screen checks x2 EXOSAP Post-PCR NX Mutation Surveyor project & archive 2-directional Sequencing set up Post-PCR NX Sequencing analysis & archive Bead clean up Post-PCR FX 3730 sequencing New BRCA Screening Workflow

  12. Result of New Screening Strategy • Cleared Backlog of 453 samples • Now screened 439 new samples in this way • Average turnaround times for new samples reduced to 23.36 days (not inc weekends) • 98.9% reported within 40 days (8 weeks)

  13. What Next? • Transfer other diseases to similar strategy • Other large gene screens • Smaller genes with relatively high number of patients

  14. New High Throughput Disease Screens

  15. Mixed and Half Plates • Introduced due to lower number of samples for some diseases eg GS, FAP, PHP • Primer spot plates with both GS and FAP primers • PHP Plates allow for half plate sequencing if sample numbers are low

  16. PHP Plate Layout

  17. Impacts on the Lab • Use of Robotics • Increases speed and accuracy of transfers • Reduces need for long checks • Reduces risk of human error • Increases screening capacity of the lab • Vast reduction in number of man hours required to perform the screening

  18. User feedback: Clinicians “It makes a lot of difference to patients (and us) to be able to give a realistic and reliable timeframe for results. In some cases it can also influence management. The new service is so quick and efficient that instead of complaining about not having results we are swamped with families to offer predictive testing to!” Dr Carole McKeown, Consultant Geneticist

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