The “Spanning Protocol”: A new DNA extraction method for efficient single-cell genetic diagnosis

Abstract

The spanning protocol was most efficient for extracting DNA from a single cell and should be particularly useful for preimplantation genetic diagnosis.

Rapid Identification of Genome-Edited Mesenchymal Stem Cell Colonies via Cas9

“…NL ( N -Lauroylsarcosine salt) method: this method was performed as described before with modifications [13]. Briefly, the cells were suspended in 15 μl lysis buffer (3 μl H 2 O, 3 μl 250 ng/μl polyadenylic acid, 3 μl 10 mM EDTA, 3 μl 250 mM DTT, 3 μl 0.5% N -Lauroylsarcosine salt solution); followed by incubation at 95°C for 10 min; the supernatant was used for PCR directly after centrifugation at 12,000 rpm for 1 min.…”

An efficient and reliable DNA extraction method for preimplantation genetic diagnosis: a comparison of allele drop out and amplification rates using different single cell lysis methods

“…For the Proteinase K/SDS lysis buffer method, the cell was placed in 3 mL lysis buffer (1 mL of 17 mM SDS and 2 mL of 125 mg/ml proteinase K), centrifuged, and incubated for 60 minutes at 37 C followed by 15 minutes at 95 C (21). Finally, for the N-lauroylsarcosine salt solution method, the cell was placed in a PCR tube containing 10 mL of DNA lysis buffer composed of H 2 O (2 mL), 250 ng/mL polyadenylic acid (2 mL), 10 mM EDTA (2 mL), 250 mM dithiothreitol solution (2 mL), and 0.5% N-lauroylsarcosine salt solution (2 mL), and stored at À20 C until the PCR assays were performed (16).…”

“…It depends on the cell type analyzed (11), the genes tested (12), the lysis conditions (13,14), as well as the PCR conditions (15). Tsuchiya et al (16) have recently developed a new DNA extraction method, called the ''Spanning protocol.'' These investigators reported that their spanning protocol decreases the incidence of amplification failure, as well as the rate of false-negatives.…”

Well-devised quantification analysis for duplication mutation of Duchenne muscular dystrophy aimed at preimplantation genetic diagnosis

“…For isolation of single lymphocytes, an aliquot of the cell suspension was thawed and 1 µl was added to 4 ml of 90% RPMI1640 supplemented with 10% FCS in a culture plate. Individual lymphocytes were deposited in 4 µl of lysis solution 1 (0.2% sarcosyl, TE containing 10 mM EDTA) [23], with a mouth-controlled glass microcapillary under an inverted microscope. The cells were then denatured at 65 • C for 10 min and kept frozen at −20 • C until the PCR procedure.…”