疟蚊多线染色体的荧光原位杂交

Ai Xia; Ashley  Peery; Maryam Kamali; Jiangtao Liang; Maria V. Sharakhova; Igor V. Sharakhov

doi:10.21769/BioProtoc.860

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Peer-reviewed

Fluorescence in situ Hybridization to the Polytene Chromosomes of Anopheles Mosquitoes

疟蚊多线染色体的荧光原位杂交

MS Maria V. Sharakhova

IS Igor V. Sharakhov email

发布: 2013年08月20日第3卷第16期 DOI: 10.21769/BioProtoc.860 浏览次数: 12708

评审: Fanglian HeYoko EguchiLin Fang

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参见作者原研究论文

The authors used this protocol in:

Cover of PLOS Pathogens, featuring study using the protocol.

Oct 2012

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Abstract

Fluorescence in situ hybridization (FISH) is a method that uses a fluorescently labeled DNA probe for mapping the position of a genetic element on chromosomes. A DNA probe is prepared by incorporating Cy-3 or Cy-5 labeled nucleotides into DNA by nick-translation or a random primed labeling method. This protocol was used to map genes (Sharakhova et al., 2010) and microsatellite markers (Kamali et al., 2011; Peery et al., 2011) on polytene chromosomes from ovarian nurse cells and salivary glands of malaria mosquitoes. Detailed physical genome mapping performed on polytene chromosomes has the potential to link DNA sequences to specific chromosomal structures such as heterochromatin (Sharakhova et al., 2010). This method also allows comparative cytogenetic studies (Sharakhova et al., 2011; Xia et al., 2010), and reconstruction of species phylogenies (Kamali et al., 2012).

Keywords: Mosquito (蚊子)

Materials and Reagents

Early fourth instar Anopheles larvae
Female Anopheles mosquitoes
Template DNA
Fisherfinest* Premium Extra-Thick Frosted Microscope Slides (Double frosted coating) (Thermo Fisher Scientific, catalog number: 12-544-6 )
Fisherfinest* Premium Cover Glasses (22 x 22 mm) (Thermo Fisher Scientific, catalog number: 12-544-10 )
50% Propionic acid in water
Razor blade
Liquid nitrogen
Ethanol, molecular biology grade
Microscope slide staining jar with lid
Random Primed DNA Labeling Kit (Roche Applied Science, catalog number: 11004760001 )
Random Primers DNA Labeling System (Life Technologies, Invitrogen^TM, catalog number: 18187-013 )
Formamide (Super pure) (Fisher Bioreagent, catalog number: BP228-100 )
Dextran Sulfate Sodium salt from Leuconostoc spp. (Sigma-Aldrich, catalog number: D8906 )
Prolong^® Gold antifade reagent (Life Technologies, Invitrogen^TM, catalog number: P36930 )
Cy3-dUTP (GE Healthcare, catalog number: PA53022 )
Cy5-dUTP (GE Healthcare, catalog number: PA55022 )
YOYO^®-1 Iodide (491/509)–1 mM Solution in DMSO (Life Technologies, Invitrogen^TM, catalog number: Y3601 )
Paraformaldehyde (Sigma-Aldrich, catalog number: F8775 )
DNA polymerase I (Fermentas, catalog number: EP0041 )
DNase I (Fermentas, catalog number: EN0521 )
QIAquick^® Gel Extraction Kit (QIAGEN, catalog number: 28704 )
QIAquick^® PCR purification Kit (QIAGEN, catalog number: 28104 )
Carnoy's solution (see Recipes)
20x SSC (see Recipes)
3 M NaAC (see Recipes)
1x PBS (see Recipes)
Hybridization buffer (see Recipes)

Equipment

1.5 ml microcentrifuge tubes
Forceps
Disposable transfer pipette
Dissecting needles
Research stereo microscope (Leica, model: VA-OM-E194-354 )
Phase contrast compound microscope with 10x, 20x, 40x and 100x objective lenses
Thermal cycler
Vacufuge^® vacuum concentrator (Eppendorf, model: 022820001 )
Incubator
Water Bath
Vortexer
Confocal Microscope or Fluorescence Microscope

Procedure

English

中文翻译

文章信息

版权信息

如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

Xia, A., Peery, A., Kamali, M., Liang, J., Sharakhova, M. V. and Sharakhov, I. V. (2013). Fluorescence in situ Hybridization to the Polytene Chromosomes of Anopheles Mosquitoes. Bio-protocol 3(16): e860. DOI: 10.21769/BioProtoc.860.
Kamali, M., Xia, A., Tu, Z. and Sharakhov, I. V. (2012). A new chromosomal phylogeny supports the repeated origin of vectorial capacity in malaria mosquitoes of the Anopheles gambiae complex. PLoS Pathog 8(10): e1002960.