|
|
|
|
| LEADER |
01000caa a22002652 4500 |
| 001 |
NLM249768038 |
| 003 |
DE-627 |
| 005 |
20250218154334.0 |
| 007 |
cr uuu---uuuuu |
| 008 |
231224s2015 xx |||||o 00| ||eng c |
| 024 |
7 |
|
|a 10.2144/000114280
|2 doi
|
| 028 |
5 |
2 |
|a pubmed25n0832.xml
|
| 035 |
|
|
|a (DE-627)NLM249768038
|
| 035 |
|
|
|a (NLM)26054765
|
| 040 |
|
|
|a DE-627
|b ger
|c DE-627
|e rakwb
|
| 041 |
|
|
|a eng
|
| 100 |
1 |
|
|a Wu, Haotian
|e verfasserin
|4 aut
|
| 245 |
1 |
0 |
|a Rapid method for the isolation of mammalian sperm DNA
|
| 264 |
|
1 |
|c 2015
|
| 336 |
|
|
|a Text
|b txt
|2 rdacontent
|
| 337 |
|
|
|a ƒaComputermedien
|b c
|2 rdamedia
|
| 338 |
|
|
|a ƒa Online-Ressource
|b cr
|2 rdacarrier
|
| 500 |
|
|
|a Date Completed 03.03.2016
|
| 500 |
|
|
|a Date Revised 03.01.2025
|
| 500 |
|
|
|a published: Electronic-eCollection
|
| 500 |
|
|
|a Citation Status MEDLINE
|
| 520 |
|
|
|a The unique DNA packaging of spermatozoa renders them resistant to DNA isolation techniques used for somatic cells, requiring alternative methods that are slow and labor intensive. Here we present a rapid method for isolating high-quality sperm DNA. Isolated human sperm cells were homogenized with 0.2 mm steel beads for 5 min at room temperature in the presence of guanidine thiocyanate lysis buffer supplemented with 50 mM tris(2-carboxyethyl)phosphine (TCEP). Our method yielded >90% high-quality DNA using 3 different commercially available silica-based spin columns. DNA yields did not differ between immediate isolation (2.84 ± 0.04 pg/cell) and isolation after 2 weeks of homogenate storage at room temperature (2.91 ± 0.13 pg/cell). DNA methylation analyses revealed similar methylation levels at both time points for three imprinted loci. Our protocol has many advantages: it is conducted at room temperature; lengthy proteinase K (ProK) digestions are eliminated; the reducing agent, TCEP, is odorless and stable at room temperature; nucleic acids are stabilized, allowing storage of homogenate; and it is adaptable for other mammalian species. Taken together, the benefits of our improved method have important implications for settings where sample processing constraints exist
|
| 650 |
|
4 |
|a Journal Article
|
| 650 |
|
4 |
|a Research Support, N.I.H., Extramural
|
| 650 |
|
4 |
|a Research Support, Non-U.S. Gov't
|
| 650 |
|
4 |
|a DNA isolation
|
| 650 |
|
4 |
|a DNA purification
|
| 650 |
|
4 |
|a epigenetics
|
| 650 |
|
4 |
|a genetics
|
| 650 |
|
4 |
|a methods
|
| 650 |
|
4 |
|a nucleic acids
|
| 650 |
|
4 |
|a sperm
|
| 650 |
|
4 |
|a spermatozoa
|
| 650 |
|
7 |
|a Phosphines
|2 NLM
|
| 650 |
|
7 |
|a Reducing Agents
|2 NLM
|
| 650 |
|
7 |
|a tris(2-carboxyethyl)phosphine
|2 NLM
|
| 650 |
|
7 |
|a H49AAM893K
|2 NLM
|
| 650 |
|
7 |
|a Silicon Dioxide
|2 NLM
|
| 650 |
|
7 |
|a 7631-86-9
|2 NLM
|
| 650 |
|
7 |
|a DNA
|2 NLM
|
| 650 |
|
7 |
|a 9007-49-2
|2 NLM
|
| 700 |
1 |
|
|a de Gannes, Matthew K
|e verfasserin
|4 aut
|
| 700 |
1 |
|
|a Luchetti, Gianna
|e verfasserin
|4 aut
|
| 700 |
1 |
|
|a Pilsner, J Richard
|e verfasserin
|4 aut
|
| 773 |
0 |
8 |
|i Enthalten in
|t BioTechniques
|d 1993
|g 58(2015), 6 vom: 13. Juni, Seite 293-300
|w (DE-627)NLM012627046
|x 1940-9818
|7 nnns
|
| 773 |
1 |
8 |
|g volume:58
|g year:2015
|g number:6
|g day:13
|g month:06
|g pages:293-300
|
| 856 |
4 |
0 |
|u http://dx.doi.org/10.2144/000114280
|3 Volltext
|
| 912 |
|
|
|a GBV_USEFLAG_A
|
| 912 |
|
|
|a SYSFLAG_A
|
| 912 |
|
|
|a GBV_NLM
|
| 912 |
|
|
|a GBV_ILN_21
|
| 912 |
|
|
|a GBV_ILN_22
|
| 912 |
|
|
|a GBV_ILN_39
|
| 912 |
|
|
|a GBV_ILN_40
|
| 912 |
|
|
|a GBV_ILN_50
|
| 912 |
|
|
|a GBV_ILN_60
|
| 912 |
|
|
|a GBV_ILN_62
|
| 912 |
|
|
|a GBV_ILN_65
|
| 912 |
|
|
|a GBV_ILN_70
|
| 912 |
|
|
|a GBV_ILN_99
|
| 912 |
|
|
|a GBV_ILN_121
|
| 912 |
|
|
|a GBV_ILN_130
|
| 912 |
|
|
|a GBV_ILN_227
|
| 912 |
|
|
|a GBV_ILN_350
|
| 912 |
|
|
|a GBV_ILN_618
|
| 912 |
|
|
|a GBV_ILN_640
|
| 912 |
|
|
|a GBV_ILN_754
|
| 912 |
|
|
|a GBV_ILN_2001
|
| 912 |
|
|
|a GBV_ILN_2002
|
| 912 |
|
|
|a GBV_ILN_2003
|
| 912 |
|
|
|a GBV_ILN_2005
|
| 912 |
|
|
|a GBV_ILN_2006
|
| 912 |
|
|
|a GBV_ILN_2007
|
| 912 |
|
|
|a GBV_ILN_2008
|
| 912 |
|
|
|a GBV_ILN_2009
|
| 912 |
|
|
|a GBV_ILN_2010
|
| 912 |
|
|
|a GBV_ILN_2012
|
| 912 |
|
|
|a GBV_ILN_2015
|
| 912 |
|
|
|a GBV_ILN_2018
|
| 912 |
|
|
|a GBV_ILN_2023
|
| 912 |
|
|
|a GBV_ILN_2035
|
| 912 |
|
|
|a GBV_ILN_2040
|
| 912 |
|
|
|a GBV_ILN_2060
|
| 912 |
|
|
|a GBV_ILN_2099
|
| 912 |
|
|
|a GBV_ILN_2105
|
| 912 |
|
|
|a GBV_ILN_2121
|
| 912 |
|
|
|a GBV_ILN_2470
|
| 951 |
|
|
|a AR
|
| 952 |
|
|
|d 58
|j 2015
|e 6
|b 13
|c 06
|h 293-300
|