Linear amplification coupled with controlled extension as a means of probe amplification in a cDNA array and gene expression analysis during cold acclimation in alfalfa (Medicago sativa L.).
This study describes a rapid and simple way to amplify limited amounts of probes used for cDNA array hybridization while maintaining the original representation of transcripts in the samples. The approach is based on linear amplification of cDNA-coupled controlled extension of amplified products and yielded a 50-75-fold increases in hybridization signal intensity. Controlled extension of products is achieved either by adjusting the amplification conditions or by using a digested template. Linear amplification with controlled extension generates a population of fragments consisting mainly of 3'-end portions of original transcripts and ranging in length from 200 to 800 nucleotides. cDNA array analysis revealed that amplified and non-amplified probes generate expression profiles with correlations ranging from r=0.857 to 0.895. Up to 90% of cDNA clones, differentially expressed during cold acclimation in alfalfa, could be detected with both types of probes. This amplification method should increase the utility of cDNA arrays for identifying novel differentially expressed genes as well as expression profiling in specialized tissues or cells when the amount of analysed material is limited. The possibility of diminishing cross-hybridization of long genes sharing high sequence homology and improving the hybridization kinetics of complex probes after amplification is also discussed.
Ivashuta
Sergey
S
Graduate School of Agriculture, Hokkaido University, North-9 West-9, Kita-ku, Sapporo 060-8589, Japan. sergey@affrc.go.jp
Uchiyama
Kazuhiro
K
Gau
Mitsuru
M
Shimamoto
Yoshiya
Y
D000064:Acclimatization / Q000235:genetics* / Q000502:physiology; D003080:Cold Temperature; D018076:DNA, Complementary / Q000235:genetics; D020869:Gene Expression Profiling / Q000379:methods*; D018506:Gene Expression Regulation, Plant; D000455:Medicago sativa / Q000235:genetics* / Q000502:physiology; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016133:Polymerase Chain Reaction
D018076:DNA, Complementary