BACKGROUND
DNA Copy Number• It was previously thought that genes are  present in two copies in the genome• Large segments of DNA can ...
Copy Number and Diseases• Predisposition to diseases• Biomarkers  – Molecular alterations specific to cancer     • Gene am...
CNVs and CNAsCopy Number Variation          Copy Number Alteration• Germline cells               • Somatic cells• Genetic ...
Obstacles and Difficulties• Expensive• Extensive bioinformatics support• Requires considerable quantities of  genomic DNA ...
Objectives• How small are micro-CNAs that can be detected?• Can they reliable detect small CNA using small  quantities of ...
METHODOLOGY
Genomic DNA Preparations           Cultured MCF-7 cell lineHarvest of cells in exponential phaseExtraction of genomic DNA ...
Array CGH and Data             analysis                        Purification by Labeled by random                          ...
Comparative Genome        Hybridization• Developed to survey CNVs in the genome• Labeled sample and reference genomic  DNA...
CGH analysis of a tumor         cell
Array CGH• BAC, P1, cosmid or cDNA clones are used  for hybridization• Microarray technology• Increased resolution
The detection of micro-CNAs usingarray-CGHRESULTS AND DISCUSSION
Detected micro-CNA                      Amplification   DeletionWith genes involved        22           15   Single genes ...
Summary of Affected Genes• Total of 84 genes with discovered CNA• Common biological process affected:  – Cell Cycle     • ...
Detection of Breakpoints ofChromosomal RearrangementsRESULTS AND DISCUSSION
Breakpoints in Chromosomal      Rearrangement• Double-stranded DNA breaks      • Translocation      • Amplification      •...
Ultradense array CGH reveals micro-amplifications and micro-deletions which areartifacts inherent to whole genome amplific...
Genome and Chromosomal           Level• Reproducible results  – Four separate experiments     • Three WGA     • One withou...
Zoom in of Chromosome 1
Zoom in of chromosome 2
Subchromosomal level• Repetitive, periodic aartifacts in amplified  samples• Wave effects   – Discreet decreases in DNA co...
CONCLUSION
Summary of Findings• Limit of sensitivity for 244 K array:      • ~100 kb• Two novel amplicons      • USP6 and PECAM1• Mic...
Comments on the Technology           Used• Cluttered 1 M microarray profile      • Wave artifacts from whole genome amplif...
Issues and Concerns• Privacy and confidentiality• Psychological impact• Clinical issues     • Highly sensitive technology ...
End of Presentation    Gomez Marineil C.
Genomics report
Genomics report
Genomics report
Genomics report
Genomics report
Genomics report
Genomics report
Genomics report
Genomics report
Genomics report
Genomics report
Genomics report
Genomics report
Genomics report
Genomics report
Genomics report
Genomics report
Genomics report
Genomics report
Genomics report
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Genomics report

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Genomics report

  1. 1. BACKGROUND
  2. 2. DNA Copy Number• It was previously thought that genes are present in two copies in the genome• Large segments of DNA can vary in copy number – Thousands to millions of base pairs• Lead to dosage imbalances
  3. 3. Copy Number and Diseases• Predisposition to diseases• Biomarkers – Molecular alterations specific to cancer • Gene amplifications • Gene deletions • Genomic rearrangement leading to gene fusion• Target of drugs and therapies
  4. 4. CNVs and CNAsCopy Number Variation Copy Number Alteration• Germline cells • Somatic cells• Genetic marker • Biomarker• Associations in diseases • Cancer studies – Autism, Glomerulonephritis, CNS disorders
  5. 5. Obstacles and Difficulties• Expensive• Extensive bioinformatics support• Requires considerable quantities of genomic DNA (>5 μg)• Not easily accessible
  6. 6. Objectives• How small are micro-CNAs that can be detected?• Can they reliable detect small CNA using small quantities of DNA from biopsy samples?• Does the amplification process introduce artifacts that can confound analysis of data?• What are the limitations of array CGH? • 244K • 1 M array
  7. 7. METHODOLOGY
  8. 8. Genomic DNA Preparations Cultured MCF-7 cell lineHarvest of cells in exponential phaseExtraction of genomic DNA using QIAmp kitIntegrity check using 250 μg for analysis w/o 60 ng for whole genome NanoDrop and AGE amplification amplification Amplification using φ29 Digestion with Alu I and DNA pol and random Rsa I primers
  9. 9. Array CGH and Data analysis Purification by Labeled by random Hybridization by Microconpriming by Cy5-dUTP probes at 65°C for 40 Centrifgation Filters, and Cy3-dUTP hrs UltracelYM-30 Feature Extraction Scan by DNA Washing Software 10.7.3.1 Microarray Scanner Identification of DNA CNV identificationAnalysis by Genome copy-number using Agilent Workbench 5.0.14 aberration using Genomic Workbench ADM-2 algorithm Database
  10. 10. Comparative Genome Hybridization• Developed to survey CNVs in the genome• Labeled sample and reference genomic DNA are co-hybridized to normal metaphase chromosomes
  11. 11. CGH analysis of a tumor cell
  12. 12. Array CGH• BAC, P1, cosmid or cDNA clones are used for hybridization• Microarray technology• Increased resolution
  13. 13. The detection of micro-CNAs usingarray-CGHRESULTS AND DISCUSSION
  14. 14. Detected micro-CNA Amplification DeletionWith genes involved 22 15 Single genes 9 5Highest # of genes 14 7 Total 24 15
  15. 15. Summary of Affected Genes• Total of 84 genes with discovered CNA• Common biological process affected: – Cell Cycle • 9 genes – Estrogen Receptor signaling • 5 genes • FOXA1: candidate biomarker of poor prognosis in breast tumors • BMP7: biomarker of bone metastasis in breast cancer • VAV3: oncogene known to be overexpressed in MCF-7 cell lines
  16. 16. Detection of Breakpoints ofChromosomal RearrangementsRESULTS AND DISCUSSION
  17. 17. Breakpoints in Chromosomal Rearrangement• Double-stranded DNA breaks • Translocation • Amplification • Deletion• Intragenic alterations in DNA copy number – Gene fusion events are more common than previously believed• Complex sequence rearrangement – Chromosomal segments break down to smaller regions which differ in copy number values – Detection due to dense spacing of probes in array
  18. 18. Ultradense array CGH reveals micro-amplifications and micro-deletions which areartifacts inherent to whole genome amplificationRESULTS AND DISCUSSION
  19. 19. Genome and Chromosomal Level• Reproducible results – Four separate experiments • Three WGA • One without amplification• No apparent change whether the DNA was amplified or not
  20. 20. Zoom in of Chromosome 1
  21. 21. Zoom in of chromosome 2
  22. 22. Subchromosomal level• Repetitive, periodic aartifacts in amplified samples• Wave effects – Discreet decreases in DNA copy number values (10- 100 kb) – Intervals of 50 – 500 kb – Hardly visible with 244 K array• Considerably confounded the analysis of ADM-2 algorithm• Does not obscure detection of true CNAs
  23. 23. CONCLUSION
  24. 24. Summary of Findings• Limit of sensitivity for 244 K array: • ~100 kb• Two novel amplicons • USP6 and PECAM1• Micro-CNAs that cut through exonic sequences may indicate potential sites of chromosomal rearrangements and translocation• Complex intragenic DNA copy number changes caused gene fusion events
  25. 25. Comments on the Technology Used• Cluttered 1 M microarray profile • Wave artifacts from whole genome amplification• Large number of artifacts limits analysis of data at subchromosomal level • Limitations on the use of ADM-2 algorithm• Requires excellent quality of genomic DNA• Highly sensitive to confounding effects of whole genome amplifications
  26. 26. Issues and Concerns• Privacy and confidentiality• Psychological impact• Clinical issues • Highly sensitive technology requires high quality data gathering procedures• Uncertainties associated with gene tests for susceptibilities and complex conditions • Genes versus the environment
  27. 27. End of Presentation Gomez Marineil C.
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