By Diponkar Banerjee (auth.), Diponkar Banerjee, Sohrab P. Shah (eds.)
The earliest descriptions of human chromosomes initiated the genomics revolution that's now upon us. Array Comparative Genomic Hybridization: Protocols and Applicationsexplores the scope of what's now attainable so far as documenting abnormalities linked to a number of human cancers. whereas the know-how for interrogating the human genome maintains to conform, refinement of comparative genomic hybridization (CGH) utilizing array CGH and similar applied sciences have supplied huge, immense perception into human cancers at a reasonable scale. Written within the winning Methods in Molecular Biology™ sequence layout, chapters comprise introductions to their respective subject matters, lists of the required fabrics and reagents, step by step, effortlessly reproducible protocols, and notes on troubleshooting and averting identified pitfalls.
Authoritative and simply obtainable, Array Comparative Genomic Hybridization: Protocols and Applicationsprovides researchers with well-honed methodologies to benefit those concepts for his or her personal use in study or medical diagnostic laboratories.
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Extra info for Array Comparative Genomic Hybridization: Protocols and Applications
At sufficient sequencing depths, on the order of two Gigabases aligned (30 million 75 nt reads; ~25% of a HiSeq2000 lane), MeDIP-seq is capable of identifying 70–80% of the 28 million CpGs in the human haploid genome at a resolution of 100–300 bases (65). This is near to the expected frequency of methylation in the human genome (17, 23). A recent comparison of published 5hmC datasets showed striking differences in enrichment profiles suggesting that additional standardization in methodologies and/ or reagents is required (66).
9. Pass the suspension in the Eppendorf tube through a 20G needle five times using a syringe to disaggregate nuclei. 10. Add 3 μl of a DAPI working solution (100×) per 300 µl of extraction buffer and incubate on ice for 3 min. 11. Filter sample through the mesh (35 μm) into the FACS tube. 12. Wash the filter with 200 μl of extraction buffer containing 1× DAPI. 3. Flow Sorting of Nuclei 1. Nuclei are sorted according to DAPI intensity using BD FACSAria flow cytometer/sorter. Excitation is achieved with violet laser (408 nm) and emission of DAPI is detected with a 450/40 filter (see Note 8).
We collected also two other, less defined subpopulations, P4 and P5. The number of nuclei in these two subpopulations was sufficient for DNA extraction (19,770 and 20,043 respectively). However, often this may not be the case and sufficient numbers of nuclei can be obtained only from one or two main subpopulations (b) Needle biopsy B-098 contained only one main diploid tumor subpopulation P6, which yielded 11,343,376 nuclei. The number of nuclei collected from P7 and P8 subpopulations was not sufficient to obtain enough DNA for array CGH analysis (9,600 and 3,891 respectively).