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A microarray approach for identifying mutated genes

Zakhrabekova, Shakhira LU ; Kannangara, C. Gamini; von Wettstein, Diter and Hansson, Mats LU (2002) In Plant Physiology and Biochemistry 40(3). p.189-197
Abstract
This study was performed to evaluate if microarray technology can identify genes using their transcriptionally defective mutant alleles. Three barley (Hordeum vulgare L.) mutant strains, xantha-h57, xantha-f27 and xantha-g28, with mutations in the genes encoding the three subunits of the chlorophyll biosynthetic enzyme magnesium chelatase, were used in a reconstruction experiment. The mutation xantha-h57 prevents transcription of Xantha-h mRNA. Microarrays were prepared by robotic spotting of PCR products from 968 clones at 1760 positions. Most of the materials were from cloned expressed sequence tags (ESTs). The barley Xantha-h gene was printed at six positions in duplicate. Crude bacterial lysates as well as purified plasmids were used... (More)
This study was performed to evaluate if microarray technology can identify genes using their transcriptionally defective mutant alleles. Three barley (Hordeum vulgare L.) mutant strains, xantha-h57, xantha-f27 and xantha-g28, with mutations in the genes encoding the three subunits of the chlorophyll biosynthetic enzyme magnesium chelatase, were used in a reconstruction experiment. The mutation xantha-h57 prevents transcription of Xantha-h mRNA. Microarrays were prepared by robotic spotting of PCR products from 968 clones at 1760 positions. Most of the materials were from cloned expressed sequence tags (ESTs). The barley Xantha-h gene was printed at six positions in duplicate. Crude bacterial lysates as well as purified plasmids were used as template in the PCR reactions. The bacterial lysates did not affect the printing quality of the DNA. This simplification is important, as it increases the throughput capacity and decreases costs. cDNA from the three mutants and the wild-type strain were differentially labeled with fluorescing nucleotides. Labeled cDNA from one mutant was mixed in equal amounts with labeled cDNA of another mutant or wild type and competitively hybridized to the microarrayed clones. The combination of labeled cDNA from xantha-h57 with that of xantha-f27 or xantha-g28 specifically highlighted the positions representing the Xantha-h gene on the microarrays. Therefore, we regard this experiment as a demonstration that microarrays provide a very promising method for screening large DNA libraries in order to clone genes known only through their mutant phenotype. This opens up a new way of using the microarray technology for cloning genes from eukaryotes with complex genomes for which genome sequencing is unlikely to proceed. Our results also put the many available plant mutant collections in focus as treasuries for gene hunters. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Barley, Cloning, Genomics, Hordeum vulgare, Magnesium chelatase, Mutant, xantha
in
Plant Physiology and Biochemistry
volume
40
issue
3
pages
189 - 197
publisher
Elsevier
external identifiers
  • wos:000174884700001
  • scopus:0036489607
ISSN
1873-2690
DOI
10.1016/S0981-9428(02)01363-3
language
English
LU publication?
yes
id
9b4eb475-2f0d-48b9-8ae0-583f4fb6aff3 (old id 124899)
date added to LUP
2007-07-06 16:52:47
date last changed
2017-10-01 04:34:52
@article{9b4eb475-2f0d-48b9-8ae0-583f4fb6aff3,
  abstract     = {This study was performed to evaluate if microarray technology can identify genes using their transcriptionally defective mutant alleles. Three barley (Hordeum vulgare L.) mutant strains, xantha-h57, xantha-f27 and xantha-g28, with mutations in the genes encoding the three subunits of the chlorophyll biosynthetic enzyme magnesium chelatase, were used in a reconstruction experiment. The mutation xantha-h57 prevents transcription of Xantha-h mRNA. Microarrays were prepared by robotic spotting of PCR products from 968 clones at 1760 positions. Most of the materials were from cloned expressed sequence tags (ESTs). The barley Xantha-h gene was printed at six positions in duplicate. Crude bacterial lysates as well as purified plasmids were used as template in the PCR reactions. The bacterial lysates did not affect the printing quality of the DNA. This simplification is important, as it increases the throughput capacity and decreases costs. cDNA from the three mutants and the wild-type strain were differentially labeled with fluorescing nucleotides. Labeled cDNA from one mutant was mixed in equal amounts with labeled cDNA of another mutant or wild type and competitively hybridized to the microarrayed clones. The combination of labeled cDNA from xantha-h57 with that of xantha-f27 or xantha-g28 specifically highlighted the positions representing the Xantha-h gene on the microarrays. Therefore, we regard this experiment as a demonstration that microarrays provide a very promising method for screening large DNA libraries in order to clone genes known only through their mutant phenotype. This opens up a new way of using the microarray technology for cloning genes from eukaryotes with complex genomes for which genome sequencing is unlikely to proceed. Our results also put the many available plant mutant collections in focus as treasuries for gene hunters.},
  author       = {Zakhrabekova, Shakhira and Kannangara, C. Gamini and von Wettstein, Diter and Hansson, Mats},
  issn         = {1873-2690},
  keyword      = {Barley,Cloning,Genomics,Hordeum vulgare,Magnesium chelatase,Mutant,xantha},
  language     = {eng},
  number       = {3},
  pages        = {189--197},
  publisher    = {Elsevier},
  series       = {Plant Physiology and Biochemistry},
  title        = {A microarray approach for identifying mutated genes},
  url          = {http://dx.doi.org/10.1016/S0981-9428(02)01363-3},
  volume       = {40},
  year         = {2002},
}