PMID- 15651009
OWN - NLM
STAT- MEDLINE
DCOM- 20050602
LR  - 20220318
IS  - 1552-4922 (Print)
IS  - 1552-4922 (Linking)
VI  - 63
IP  - 2
DP  - 2005 Feb
TI  - A novel approach to prepare extended DNA fibers in plants.
PG  - 114-7
AB  - BACKGROUND: The extended DNA fiber preparation procedure is still imperfect in 
      plants due to the existence of a hard cell wall; thus, high quality of extended 
      DNA fibers for fluorescence in situ hybridization (FISH) analysis is often 
      difficult to be obtained rapidly and efficiently. In this study we have developed 
      a fast and widely effective method to prepare DNA fibers from various plant 
      species and the fibers are suitable for fiber FISH mapping. METHODS: Fresh young 
      leaves were chopped with a sharp sterile scalpel in a Petri dish that contained 
      ice-cold nucleus isolation buffer followed by filtration through 33-mum nylon 
      mesh. Nuclei were obtained by centrifuging the filtrates at high speed (16,000g) 
      for 40 s. Nucleus lysis buffer (0.5% sodium dodecylsulfate, 5 mM 
      ethylenediaminetetraacetic acid, 100 mM Tris, pH7.0) was added to nuclei on 
      slides, and DNA fibers were dragged and extended with a clean coverslip. RESULTS: 
      The key of this method is that liquid nitrogen grinding of leaves is replaced by 
      chopping with a blade in ice-cold nucleus isolation buffer. With the liquid 
      nitrogen method, over- or under-grinding of leaves occurs more frequently, and 
      DNA fibers with the desired quality are not obtained easily. In contrast, it is 
      easier to release nuclei from cells in nucleus isolation buffer by chopping, 
      which results in fewer nuclei being destroyed. Highly extended, intact, and long 
      DNA fibers can be generated to a great probability with this method. In addition, 
      this method is very simple and rapid, requiring only 20 min for the entire 
      process, and is also safe because poisonous mercaptoethanol is replaced by 
      dithiothreitol. The results of fiber-FISH with maize genomic DNA and 45S rDNA as 
      probes showed that DNA fiber size as long as 1.96 Mb could be measured. The 
      successful and reliable preparation of maize, wild rice, and barley DNA fibers 
      suitable for FISH mapping proves that this technique is a widely effective 
      approach for obtaining extended DNA fibers in plants. CONCLUSIONS: A simple, 
      rapid, safe, and widely effective method for getting extended DNA fibers has been 
      developed in plants. (c) 2005 Wiley-Liss, Inc.
FAU - Li, Lijia
AU  - Li L
AD  - Key Laboratory of MOE for Plant Developmental Biology, College of Life Sciences, 
      Wuhan University, Wuhan 430 072, China. lljia88@yahoo.com.cn
FAU - Yang, Jinling
AU  - Yang J
FAU - Tong, Qiong
AU  - Tong Q
FAU - Zhao, Lijuan
AU  - Zhao L
FAU - Song, Yunchun
AU  - Song Y
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - United States
TA  - Cytometry A
JT  - Cytometry. Part A : the journal of the International Society for Analytical 
      Cytology
JID - 101235694
RN  - 0 (Chromatin)
RN  - 0 (DNA, Plant)
SB  - IM
MH  - Chromatin/chemistry/isolation & purification
MH  - DNA, Plant/*chemistry/isolation & purification
MH  - Hordeum/chemistry
MH  - In Situ Hybridization, Fluorescence
MH  - Oryza/chemistry
MH  - Plant Leaves/chemistry
MH  - Poaceae/chemistry/*genetics
MH  - Zea mays/chemistry
EDAT- 2005/01/15 09:00
MHDA- 2005/06/03 09:00
CRDT- 2005/01/15 09:00
PHST- 2005/01/15 09:00 [pubmed]
PHST- 2005/06/03 09:00 [medline]
PHST- 2005/01/15 09:00 [entrez]
AID - 10.1002/cyto.a.20111 [doi]
PST - ppublish
SO  - Cytometry A. 2005 Feb;63(2):114-7. doi: 10.1002/cyto.a.20111.