β-mercaptoethanol assists efficient construction of sperm bacterial artificial chromosome library

Abstract

Bacterial artificial chromosome (BAC) library plays a critical role in the strategic research in genomics. Sperm is known as a good source for BAC library construction. However, preparation of intact DNA from the highly condensed sperm nuclei is not easy. Here we developed and validated an efficient DNA extraction strategy for BAC library construction from sperm embedded in agarose plugs. The protocol used a combination of lauroylsarcosine, proteinase K and β-mercaptoethanol (a reducing agent of nucleus) In comparison with the normal protocol without reducing agents, β-mercaptoethanol released high-molecular-weight DNA from the protamines which permit DNA to be packed very densely within the spermatozoan nucleus, without damaging DNA. Extracted DNA by this method was readily digested by restriction enzymes and ideal for BAC library construction.

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“…Lysates were then mixed with 6 M Guanidinium Thiocyanate (GITC), SPRI beads suspended in 1x TE buffer (10 mM Tris pH 8.0, 1 mM EDTA), and isopropanol in a ratio of 1:2:2:4, respectively. For DNA extraction from sperm, the frozen pellet (described above) was resuspended in GITC supplemented with 10% v/v ß-mercaptoethanol as a reducing agent 85 . For all tissues, after a 5 min incubation to allow DNA to bind to the magnetic beads, tubes were placed on a neodymium magnetic rack for ~5 min until the solution was clarified.…”