RESUMO
We overexpressed duplex-specific nuclease (DSN) from Kamchatka crab in Escherichia coli cells and developed procedures for purification, renaturation, and activation of this protein. We demonstrated identity of the properties of the native and recombinant DSN. We also successfully applied the recombinant DSN for full-length cDNA library normalization.
Assuntos
Braquiúros/enzimologia , Endonucleases/metabolismo , Proteínas Recombinantes/metabolismo , Animais , Sequência de Bases , Braquiúros/genética , Braquiúros/metabolismo , Clonagem Molecular/métodos , DNA Complementar/genética , Endonucleases/química , Endonucleases/genética , Ativação Enzimática , Estabilidade Enzimática , Ácidos Nucleicos Heteroduplexes/metabolismo , Dobramento de Proteína , Renaturação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
A new modification of the single nucleotide polymorphism (SNP) analysis (DSNP, duplex-specific nuclease preference) method using the duplex-specific nuclease from the king crab was proposed. The method was used to study SNPs in the following human genes: kRAS, nRAS, hRAS, and p53, the genes of blood coagulation factor V, methyltetrahydrofolate reductase, prothrombin, and apolipoprotein E and a deletion in the BRCA1 gene. DSNP was shown to be useful for the estimation of the mutant allele content in DNA samples. A system for the simultaneous identification of several adjacent single-nucleotide polymorphisms in the kRAS gene was proposed. The approaches could be used to develop test systems for the detection of SNPs in human genes. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2005, vol. 31, no. 6; see also http://www.maik.ru.
Assuntos
DNA/química , Endonucleases/química , Genoma Humano , Polimorfismo de Nucleotídeo Único , Alelos , Animais , Braquiúros/enzimologia , Primers do DNA , Genes ras , Doenças Genéticas Inatas/genética , Humanos , Mutação PuntualRESUMO
We developed a new method for the preparation of normalized cDNA libraries enriched with full-length sequences. It is based on the properties of the recently characterized duplex-specific nuclease from the hepatopancreas of the Kamchatka crab. The duplex-specific nuclease is thermostable, it effectively cleaves double-stranded DNA and is inactive toward single-stranded DNA (Shagin et al., Genome Res., 2002, vol. 12, pp. 1935-1942). Our method enables the normalization of cDNA samples enriched with full-length sequences without use of laborious and ineffective stages of physical separation. The efficiency of the method was demonstrated in model experiments using cDNA samples from several human tissues.
Assuntos
DNA Complementar/química , DNA de Cadeia Simples/química , Biblioteca Gênica , Animais , Braquiúros , Endonucleases/química , Humanos , Hibridização de Ácido Nucleico , Reação em Cadeia da PolimeraseRESUMO
A transcript containing the long terminal repeat (LTR) and the sequence homologous to the KIAA1245 mRNA fragment were revealed among the transcribed LTRs of human endogenous viruses of the K family in normal and tumor tissues. Ten other sequences with a high level of homology to the KIAA1245 mRNA were found in the GenBank. The intron-exon structures were determined for all the sequences, and their exon sequences were compared. The comparison showed that they differ both in the extent of the exon homology and in the presence or absence of the HERV-K LTR in the third intron. The revealed sequences form a new gene family that comprises at least four subfamilies. Two of these subfamilies have the LTR, and the other two do not. We showed by PCR that the LTR was integrated into the introns after the divergence of the orangutan evolutionary branch from other hominoids but before the divergence of the gorilla branch, i.e., 8-13 million years ago. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2002, vol. 28, no. 4; see also http://www.maik.ru.
