Rapid sex determination on buccal smears using DNA probes and fluorescence in situ hybridization

Title: Rapid sex determination on buccal smears using DNA probes and fluorescence in situ hybridization

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Abstract

Hybridization of dual-labeled DNA probes for the repetitive sequences on the X and Y chromosomes allows a fast, non-invasive, more reliable method for sex determination that current cytogenetic Barr body and Y chromatin assays. Scrapes of squamous epithelial cells were collected from the oral cavity of 14 subjects (5{male}, 9{female}) and smeared onto silanized slides. The smears were allowed to air dry. Samples were blinded and then fixed in 50% methanol/50% glacial acetic acid for 10 minutes, and allowed to dry. The slides were incubated in a pretreatment solution containing 30% sodium bisulfite at 45{degrees}C for 10 minutes. They were rinsed in 2XSSC pH 7.0 and then dehydrated through a series of 70%, 85%, and 100% ethanols at room temperature and allowed to air dry. A probe mixture (30 {mu}L containing 10 ng/{mu}L biotin-labeled DXZ1 and digoxigenin-labeled DYZ1/DYZ3 in 70% Formamide/2XSSC) was aliquoted onto each slide, coverslipped, and sealed with rubber cement. Probe and target DNA were simultaneously denatured at 72{degrees}C on a slide warmer for 6 minutes. Probe was allowed to hybridize overnight in a humidified chamber at 37{degrees}C. Slides were postwashed at 72{degrees}C in 0.5xSSC pH 7.0 for 5 minutes, then soaked at room temperature 1XPBD for 2more »« less

Authors:

Giraldez, R A ^[1]; Harris, C ^[2]

Oncor, Inc., Gaithersburg, MD (United States)
Univ. of Wisconsin, WI (United States)

Publication Date:: 1994-09-01

OSTI Identifier:: 133685

Report Number(s):: CONF-941009-
Journal ID: AJHGAG; ISSN 0002-9297; TRN: 95:005313-0415

Resource Type:: Journal Article

Journal Name:: American Journal of Human Genetics

Additional Journal Information:: Journal Volume: 55; Journal Issue: Suppl.3; Conference: 44. annual meeting of the American Society of Human Genetics, Montreal (Canada), 18-22 Oct 1994; Other Information: PBD: Sep 1994

Country of Publication:: United States

Language:: English

Subject:: 55 BIOLOGY AND MEDICINE, BASIC STUDIES; HETEROCHROMOSOMES; DNA SEQUENCING; SEX; DIAGNOSIS; PROBES; FLUORESCENCE; DNA HYBRIDIZATION

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                    Giraldez, R A, and Harris, C. Rapid sex determination on buccal smears using DNA probes and fluorescence in situ hybridization.  United States: N. p., 1994. 
        Web.

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                    Giraldez, R A, & Harris, C. Rapid sex determination on buccal smears using DNA probes and fluorescence in situ hybridization.  United States.

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                    Giraldez, R A, and Harris, C. Thu .  
        "Rapid sex determination on buccal smears using DNA probes and fluorescence in situ hybridization".  United States.

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@article{osti_133685,

  title        = {Rapid sex determination on buccal smears using DNA probes and fluorescence in situ hybridization},

  author       = {Giraldez, R A and Harris, C},

  abstractNote = {Hybridization of dual-labeled DNA probes for the repetitive sequences on the X and Y chromosomes allows a fast, non-invasive, more reliable method for sex determination that current cytogenetic Barr body and Y chromatin assays. Scrapes of squamous epithelial cells were collected from the oral cavity of 14 subjects (5{male}, 9{female}) and smeared onto silanized slides. The smears were allowed to air dry. Samples were blinded and then fixed in 50% methanol/50% glacial acetic acid for 10 minutes, and allowed to dry. The slides were incubated in a pretreatment solution containing 30% sodium bisulfite at 45{degrees}C for 10 minutes. They were rinsed in 2XSSC pH 7.0 and then dehydrated through a series of 70%, 85%, and 100% ethanols at room temperature and allowed to air dry. A probe mixture (30 {mu}L containing 10 ng/{mu}L biotin-labeled DXZ1 and digoxigenin-labeled DYZ1/DYZ3 in 70% Formamide/2XSSC) was aliquoted onto each slide, coverslipped, and sealed with rubber cement. Probe and target DNA were simultaneously denatured at 72{degrees}C on a slide warmer for 6 minutes. Probe was allowed to hybridize overnight in a humidified chamber at 37{degrees}C. Slides were postwashed at 72{degrees}C in 0.5xSSC pH 7.0 for 5 minutes, then soaked at room temperature 1XPBD for 2 minutes, and detected with rhodamine/anti-digoxigenin-FITC/avidin for 15 minutes at 37{degrees}C. Slides were soaked 3X in 1XPBD and then counterstained with 15 {mu}L 0.05 {mu}g/mL DAP1/Antifade. 200 nuclei were scored for the presence of one green (X), two green (XX), one green and one red (XY), or a single red (Y) signal, using a fluorescent microscope equipped with a triple band pass filter. Greater than 90% of the hybridized nuclei from each of the 14 cases studied conformed to the sex chromosome pattern. The modal number in 9 cases showed two green signals (XX), and a green and a red signal (XY) in the other 5 cases; this was in complete agreement with the cytogenetic results.},

  doi          = {},

  url          = {https://www.osti.gov/biblio/133685},
  journal      = {American Journal of Human Genetics},
number       = Suppl.3,

  volume       = 55,

  place        = {United States},

  year         = {1994},

  month        = {9}

}

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