用于聚合酶链式反应(PCR)引物设计的计算机程序

 本文报道了两个用于PCR引物设计的计算机程序PCRDESN和PCRDESNA。PCRDESN程序主要从以下4个方面评价用户自己设计的一对引物的质量:(1)引物内的碱基反向重复或发夹结构,(2)两个引物之间的碱基互补配对,(3)两个引物之间的同源性,(4)引物的碱基组成及特点和T_m值计算。通过用多例文献发表的及本院有关实验室提供的引物对序列的验证,确定了程序的运算参数,证明该程序能较好地检验引物对的质量和解释某些PCR实验失败的原因。PCRDESNA程序采用逐级优化的方法和比PCRDESN所选用的更严紧的引物选择参数对用户提供的核酸序列进行快速检索,以确定所有可能的和合适的引物对。     

Construction and characterisation of a yeast artificial chromosome library containing three haploid maize genome equivalents

We have constructed a yeast artificial chromosome (YAC) library using high-molecular-weight DNA prepared from agarose-embedded leaf protoplasts of the maize inbred line UE95. This library contains 79 000 clones with an average insert size of 145 kb and should therefore represent approximately three haploid genome equivalents. The library is organised as an ordered array in duplicate microtitre plates. Forty-one pools of DNA from 1920 individual clones have been prepared for rapid screening of the library by the polymerase chain reaction (PCR). Using this approach, together with conventional colony hybridisation, we have been able to identify between one and eight positive clones for every probe used.     

Mitochondrial rps14 is a transcribed and edited pseudogene in Arabidopsis thaliana

We have isolated and analysed a 2 kb region of the mitochondrial genome of Arabidopsis thaliana (Columbia) showing a high level of nucleotide identity with the mitochondrial (mt) rps14 small-subunit ribosomal protein gene from Oenothera berteriana and Vicia faba, as well as with an open reading frame (ORF) located upstream of the nad3 locus in O. berteriana. The rps14 locus is present as a single copy in the A. thaliana mt genome and has a translational stop codon located near the initiation codon, as well as a deletion of one nucleotide that disturbs the coding sequence. The cloning and sequencing of nine amplified mt rps14 cDNAs clearly demonstrated that this gene is transcribed and that the mRNA precursors are edited at three positions, all involving C-to-U conversions. No editing events changing the stop codon and restoring the correct coding sequence were witnessed within the 9 individual cDNA clones. Therefore, we conclude that the single rps14 sequence of the mitochondrial genome from A. thealiana is in fact a pseudogene that is transcribed and edited but not translated.     

A rapid procedure for the construction of PCR cDNA libraries from small amounts of plant tissue

We describe a general method for the preparation of λZAP II cDNA libraries from very small amounts (<50 mg) of plant tissue. We have achieved this by combining an efficient method for RNA extraction with a modified PCR protocol for the synthesis and amplification of cDNA. Using this protocol we have found it possible to generate cDNA libraries containing more than 10⁶ clones from as little as 1 μg of total RNA.     

Characterization of six microsatellite markers in Trillium camschatcense using a dual‐suppression‐polymerase chain reaction technique

Trillium camschatcense is a herbaceous perennial plant distributed in Hokkaido and northern Honshu, Japan. Geographical variations in the breeding system (partial selfing or obligate outcrossing) are reported in the populations of Hokkaido. We isolated six polymorphic microsatellite loci from this species. The number of allele per locus ranged from four to 12, whereas the expected heterozygosities ranged from 0.69 to 0.83. These markers may allow further investigations to reveal the evolutionary and ecological processes of mating system in T. camschatcense.