A simple and novel DNA combing methodology for Fiber-FISH and optical mapping

• Published in: Genomics (San Diego, Calif.) Calif.), 2019-07, Vol.111 (4), p.567-578
• Main Authors: Yadav, Hemendra, Sharma, Pulkit
• Format: Article
• Language: English
• Subjects: DNA combing; DNA denaturation; DNA, Single-Stranded - chemistry; DNA, Single-Stranded - ultrastructure; Fluorescence microscopy; Humans; In Situ Hybridization, Fluorescence - methods; Nucleic Acid Denaturation; Restriction digestion; Single molecule analysis; Single Molecule Imaging - methods; Slide silanization
• ISSN: 0888-7543; 1089-8646
• DOI: 10.1016/j.ygeno.2018.03.012

Single molecule analysis can help us study genomics efficiently. It involves studying single DNA molecules for genomic studies. DNA combing is one of such techniques which allowed us to study single DNA molecules for multiple uses. DNA combing technology can be used to perform Fiber-FISH and optical mapping. Physical mapping of genomes can be studied by restriction digestion of combed DNA on glass slides. Restriction fragments can be arranged into optical maps by gathering fluorescent intensity data by CCD camera and image analysis by softwares. Physical mapping and DNA segment rearrangements can be studied by Fiber-FISH which involves application of probes on genomic DNA combed over glass slides. We developed a novel methodology involving combing solution optimization, denatured combed DNA and performed restriction digestion of combed DNA. Thus we provided an efficient and robust combing platform for its application in Fiber-FISH and optical mapping. •DNA combing methodology was optimized including slide surface coating, combing solution and method of combing.•Combed DNA was denatured, treated with ssb protein, denaturation was studied by observing fluorescent intensity.•Restriction digestion of combed DNA was performed.