Modified guanidinium thiocyanate method for human sperm DNA isolation

Abstract

Mammalian sperm chromatin is highly condensed, so isolating DNA from such chromatin can be a formidable task. The procedures that produce high quality DNA from somatic cells fail to yield quality sperm DNA. In this study we have modified the previously used guanidinium method to make it simple and efficient in isolating human sperm DNA. In our method, the lysis buffer contained guanidinium, sodium citrate, sarkosyl, proteinase K and mercaptoethanol. Proteinase K was not used in the original guanidinium method but was included in our protocol. CsCl centrifugation of the lysate, as described in the original procedure, was omitted. Instead, isopropyl alcohol was added directly to the lysis buffer to harvest the DNA. This modified guanidinium method generated high molecular weight DNA while the other two methods resulted in considerable DNA degradation. There was no difficulty in restriction enzyme digestion of DNA prepared by the modified method as revealed by Southern blot analysis. Since the modified guanidinium method is a simple one-step procedure which avoids homogenization, organic solvents, centrifugation and, more importantly, produces degradation-free DNA, it could be the method of choice when DNA from mature germ cells is needed.

A protocol for direct and rapid multiplex PCR amplification on forensically relevant samples

“…Due to their structure, sperm cells may need a more stringent protocol (longer 98 8C hotstart period) to release DNA [15]. Two semen samples were diluted so that approximately 100 cells were added to the amplification reactions.…”

Comparative study of DNA extraction methodologies from goat sperm and its effects on polymerase chain reaction analysis

“…However, it is clear that the use of the DNeasy Blood & Tissue Kit is limited by its high cost (Bailes et al, 2007), and extraction with phenol-chloroform is a laborious and potentially hazardous method (Sepp et al, 1994), where a significant amount of DNA can be lost (Goldenberger et al, 1995) and degraded (Hossain et al, 1997) and the PCR inhibited (Goldenberger et al, 1995). By contrast, Chelex-100 resin emerged as a cheap, effective, fast, and simple method, which can be realized in few steps, and does not require the use of organic solvents, which confirms observations of previous studies (Walsh et al, 1991;Simonato et al, 2007).…”

“…However, mammalian tissues exhibit considerable variation in their structures; therefore, one DNA extraction technique might not be suitable for all tissue types (Hossain et al, 1997). Sperm cells present a high degree of nuclear compaction by protamines (Griffin, 2013) and a strong nuclear membrane (Horsman et al, 2005), which inhibits the extraction of sperm DNA by standard techniques used for somatic cells (Hossain et al, 1997;Griffin, 2013), as observed in goat sperm DNA extractions (Silva et al, 2011).…”

“…Sperm cells present a high degree of nuclear compaction by protamines (Griffin, 2013) and a strong nuclear membrane (Horsman et al, 2005), which inhibits the extraction of sperm DNA by standard techniques used for somatic cells (Hossain et al, 1997;Griffin, 2013), as observed in goat sperm DNA extractions (Silva et al, 2011). Thus, the aim of this study was to evaluate three methods to extract goat sperm DNA that can be used successfully in PCR.…”

Characterization of Exon 3 of PROP1 gene and Screening of H173R polymorphism in Karan Fries bulls

“…The DNA isolation was performed using a protocol that involves two steps: Lysis and extraction. Lysis of spermatozoa was done as per Hossain et al (1997), while the extraction of DNA was done by standard Phenol Chloroform extraction procedure (Sambrook and Russell, 2001). The concentrations of DNA were measured using Biospec-nano-spectrophotometer (Schimadzu Cooperation, Japan).…”