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תפוקה גבוהה מיפוי פיזי של הכרומוזומים באמצעות אוטומטי doi: 10.3791/4007 Published: June 28, 2012

Materials

Name Company Catalog Number Comments
22x22 mm microscope coverslips Fisher 12-544-10
18x18 mm microscope coverslips Fisher 12-553-402
Acetic acid Fisher A491-212
Methanol Fisher A412-4
Propionic acid Sigma-Aldrich 402907
Dremel 200 rotary tool with a Flex-Shaft attachment Rand 3" Multi-purpose Mini Bench Grinder (with rate limiter)
Specially designed 200 μl plastic tips (beaded plastic edges) for Dremel tool Pipetman F171300 Cut and heat fat end of pipet tips
Tin coated rapid vise Avenger Gold Toolmaker MTC-200-1 Precision ground square and parallel to within 0.00025
Torque wrench Craftsman 44593
ThermoBrite Slide Denaturation/Hybridization System Abbott Molecular 30-144110
25 mm barrier slides Abbott Molecular XT108-SL
25 mm coverslips Abbott Molecular XT122-90X
Xmatrx Automated Slide Staining System Abbott Molecular 08L46-001
MZ6 Leica stereomicroscope Leica VA-OM-E194-354 A different stereomicroscope can be used
Olympus CX41 Phase Microscope Olympus CX41RF-5 A different phase microscope can be used
ACCORD PLUS Automated Scanning System BioView (USA) BV-5000-ACCP
10x PBS Invitrogen P5493
10% NBF (neutral buffered formalin) Sigma-Aldrich HT501128
99% formamide Fisher Scientific BP227500
Dextran sulfate sodium salt Sigma D8906
20x SSC buffer Invitrogen AM9765
Sodium phosphate Sigma-Aldrich S3264
50x Denhardt's solution Sigma-Aldrich D2532
Sodium azide Sigma-Aldrich S2002
1 mM YOYO-1 iodide (491/509) solution in DMSO Invitrogen Y3601
ProLong Gold antifade reagent Invitrogen P36930
dATP, dCTP, dGTP, dTTP Fermentas R0141, R0151, R0161, R0171
Cy3-dUTP, Cy5-dUTP GE Healthcare PA53022, PA55022
BSA Sigma A3294
DNA polymerase I Fermentas EP0041
DNase I Fermentas EN0521

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References

  1. Genome 10K: a proposal to obtain whole-genome sequence for 10,000 vertebrate species. J. Hered. 100, 659-674 (2009).
  2. Robinson, G. E., et al. Creating a Buzz About Insect Genomes. Science. 331, 1386-1386 (2011).
  3. Project description at Broad Institute [Internet]. The Eli and Edythe L. Broad Institute of Harvard and MIT. Cambridge, MA. Available from: http://www.broadinstitute.org/annotation/genome/anopheles/ (2012).
  4. Besansky, N. Genome Analysis Of Vectorial Capacity In Major Anopheles Vectors Of Malaria Parasites. White Paper. 1-15 (2008).
  5. Alkan, C., Sajjadian, S., Eichler, E. E. Limitations of next-generation genome sequence assembly. Nature. 8, 61-65 (2011).
  6. Sharakhova, M. V., et al. Update of the Anopheles gambiae PEST genome assembly. Genome Biol. 8, R5 (2007).
  7. Lewin, H. A., Larkin, D. M., Pontius, J., O'Brien, S. J. Every genome sequence needs a good map. Genome Res. 19, 1925-1928 (2009).
  8. Bhutkar, A., et al. Chromosomal rearrangement inferred from comparisons of 12 Drosophila genomes. Genetics. 179, 1657-1680 (2008).
  9. Novikov, D., Kireev, I., Belmont, A. S. High-pressure treatment of polytene chromosomes improves structural resolution. Nature. 4, 483-485 (2007).
  10. George, P., Sharakhova, M. V., Sharakhov, I. V. High-resolution cytogenetic map for the African malaria vector Anopheles gambiae. Insect Mol. Biol. 19, 675-682 (2010).
  11. FISH Complete Automation [Internet]. Abbott Laboratories. Abbott Park, IL. Available from: http://www.abbottmolecular.com/products/instrumentation-automation/fish/fish-automation.html (2012).
  12. The ACCORD system [Internet]. BioView Ltd. Billerica, MA. Available from: http://www.bioview.co.il/htmls/article.aspx?c0=12510&bsp=12332&bss53=12332 (2012).
  13. Clements, A. N. The Biology of Mosquitoes: Development, Nutrition and Reproduction. Chapman & Hall. (1992).
  14. BLAST @ VectorBase.org [Internet]. VectorBase.org. Notre Dame, IN. Available from: http://agambiae.vectorbase.org/Tools/BLAST/ (2012).
  15. Sharakhova, M. V., et al. Imaginal discs--a new source of chromosomes for genome mapping of the yellow fever mosquito Aedes aegypti. PLoS Negl. Trop. Dis. 5, e10 (2011).
  16. Scriven, P. N., Kirby, T. L., Ogilvie, C. M. FISH for Pre-implantation Genetic Diagnosis. J. Vis. Exp. (48), e2570 (2011).
  17. Cai, W., Jin, Y., Girton, J., Johansen, J., Johansen, K. M. Preparation of Drosophila Polytene Chromosome Squashes for Antibody Labeling. J. Vis. Exp. (36), e1748 (2010).
  18. Vatolina, T. Y., et al. Correction: Identical Functional Organization of Nonpolytene and Polytene Chromosomes in Drosophila melanogaster. PLoS ONE. 6, (2011).
תפוקה גבוהה מיפוי פיזי של הכרומוזומים באמצעות אוטומטי<em&gt; באתרו</em&gt; הכלאה
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Cite this Article

George, P., Sharakhova, M. V., Sharakhov, I. V. High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization. J. Vis. Exp. (64), e4007, doi:10.3791/4007 (2012).More

George, P., Sharakhova, M. V., Sharakhov, I. V. High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization. J. Vis. Exp. (64), e4007, doi:10.3791/4007 (2012).

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