• Title/Summary/Keyword: unclonable DNA

Search Result 2, Processing Time 0.017 seconds

Cloning and DNA Sequencing for Unstable Minisatellites DNA Regions in E. coli. (대장균 내에서 불안정한 Minisatellite DNA 영역의 클론닝 및 DNA 염기서열 결정)

  • 임선희;김재우;김광섭;정윤희;윤세련;배호정;안태진;선우양일
    • Korean Journal of Microbiology
    • /
    • v.40 no.2
    • /
    • pp.65-72
    • /
    • 2004
  • Instability of some eukaryotic sequence propagated in prokaryotic hosts is a frequently observed phenomenon. It is well documented that long inverted repeats, AT-rich sequences with structures like Z-DNA are extremely unstable in E. coli. These sequences may either be under-represented or even lost when cloned in E. coli. When we analyzed the polymorphic pattern for several tandom repeat (TR) in human SCKI gene, we found some TR regions were frequently deleted from plasmids and had difficult problem for their sequencing. These regions may result in non-clonability of the DNA sequence. Here we have cloned two difficult TR regions under low temperature and made two library for DNA sequencing using a nebulizer or sonicator. This study will help to determine the unstable genomic elements in complex mammalian genome.

In Vivo Excision and Amplification of Large Human Genomic Segments Using Cre/loxP-and EBNA-1/oriP-mediated Machinery

  • Yoon, Young-Geol;Choi, Ja-Young;Kim, Jung-Min;Lee, Jun-Hyoung;Kim, Sun-Chang
    • BMB Reports
    • /
    • v.34 no.4
    • /
    • pp.322-328
    • /
    • 2001
  • Excision and amplification of pre-determined, large genomic segments (taken directly from the genome of a natural host, which provides an alternative to conventional cloning in foreign vectors and hosts) was explored in human cells. In this approach, we devised a procedure for excising a large segment of human genomic DNA, the iNOS gene, by using the Cre/loxP system of bacteriophage P1 and amplifying the excised circles with the EBNA-1/oriP system of the Epstein-Barr virus. Two loxP sequences, each of which serves as a recognition site for recombinase Cre, were integrated unidirectionally into the 5'-UTR and 3'-UTR regions of the iNOS gene, together with an oriP sequence for conditional replication. The traps-acting genes cre and EBNA-1, which were under the control of a tetracycline responsive $P_{hcmv^*-1}$ promoter, were also inserted into the 5'-UTR and 3'-UTR regions of the iNOS gene, respectively, by homologous recombination. The strain carrying the inserted elements was stably maintained until the excision and amplification functions were triggered by the induction of cre and EBNA-1. Upon induction by doxycycline, Cre excised the iNOS gene that was flanked by two ZoxP sites and circularized it. The circularized iNOS gene was then amplified by the EBNA-1/oriP-system. With this procedure, approximately a 45.8-kb iNOS genomic fragment of human chromosome 17 was excised and successfully amplified in human cells. Our procedure can be used effectively for the sequencing of unclonable genes, the functional analysis of unknown genes, and gene therapy.

  • PDF