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Huimin Kong

Publications and source records attributed to Huimin Kong.

6 recordsLinked to original sources
PubMed2006

Simultaneous amplification and screening of whole plasmids using the T7 bacteriophage replisome.

This study describes a novel helicase-mediated isothermal DNA amplification method that exponentially amplifies circular DNAs. The circular helicase-dependent amplification (cHDA) system is based on the T7 replication machinery, which includes the processive T7 helicase, an exonuclease-deficient T7 DNA polymerase (T7 Sequenase) and the T7 Gp2.5 single-stranded DNA-binding (SSB) protein. After the duplex DNA template is unwound by the T7 helicase, specific primers anneal to the separated DNA strands and T7 Sequenase extends the 3' end of each primer by a rolling circle mechanism to amplify not only a region defined by the primers but also continuous concatemers of the template. The cHDA reaction can be carried out at one temperature (25 degrees C) for the entire process and can achieve up to 10 000-fold amplification. Amplification can be performed using purified plasmid DNA or a crude cell lysate and can amplify inserts as large as 10 kb. Following a cHDA reaction, the amplified products can be used directly for sequencing and restriction enzyme digestion without further purification. By utilizing the helicase enzyme, circular DNA samples can be simultaneously screened and amplified at one constant temperature in one easy step.

Bacteriophage T7↗
PubMed2005

Characterization of a thermostable UvrD helicase and its participation in helicase-dependent amplification.

Helicase-dependent amplification (HDA) is an isothermal in vitro DNA amplification method based upon the coordinated actions of helicases to separate double-stranded DNA and DNA polymerases to synthesize DNA. Previously, a mesophilic form of HDA (mHDA) utilizing the Escherichia coli UvrD helicase, DNA polymerase I Klenow fragment, two accessory proteins, MutL and single-stranded DNA-binding protein (SSB), was developed (1). In an effort to improve the specificity and performance of HDA, we have cloned and purified a thermostable UvrD helicase (Tte-UvrD) and the mutL homolog (Tte-MutL) from Thermoanaerobacter tengcongensis. Characterization of the Tte-UvrD helicase shows that it is stable and active from 45 to 65 degrees C. We have found that the Tte-UvrD helicase unwinds blunt-ended DNA duplexes as well as substrates possessing 3'- or 5'-ssDNA tails. Tte-UvrD was used to develop athermophilichelicase-dependent amplification (tHDA) system to selectively amplify target sequences at 60-65 degrees C. The tHDA system is more efficient than mHDA, displaying heightened amplification sensitivity without the need for the MutL and SSB accessory proteins. Amplification independent of MutL corresponds with studies demonstrating that maximal Tte-UvrD helicase activity does not require the mutL homolog. The tHDA system allows for rapid amplification and detection of targets present in genomic DNA. The expeditious nature and simplistic design of the tHDA platform makes the technology ideal for use in diagnostic applications requiring rapid identification of organisms at the point-of-need.

Adenosine Triphosphatases↗
PubMed2005

Complete genome sequence of bacteriophage T5.

The 121,752-bp genome sequence of bacteriophage T5 was determined; the linear, double-stranded DNA is nicked in one of the strands and has large direct terminal repeats of 10,139 bp (8.3%) at both ends. The genome structure is consistently arranged according to its lytic life cycle. Of the 168 potential open reading frames (ORFs), 61 were annotated; these annotated ORFs are mainly enzymes involved in phage DNA replication, repair, and nucleotide metabolism. At least five endonucleases that believed to help inducing nicks in T5 genomic DNA, and a DNA ligase gene was found to be split into two separate ORFs. Analysis of T5 early promoters suggests a probable motif AAA{3, 4 T}nTTGCTT{17, 18 n}TATAATA{12, 13 W}{10 R} for strong promoters that may strengthen the step modification of host RNA polymerase, and thus control transcription of phage DNA. The distinct protein domain profile and a mosaic genome structure suggest an origin from the common genetic pool.

Bacteriophages↗
PubMed2005

Sau42I, a BcgI-like restriction-modification system encoded by the Staphylococcus aureus quadruple-converting phage Phi42.

The serotype F phage Phi42 of Staphylococcus aureus is a triple-converting bacteriophage that encodes the staphylokinase gene (sak) and the enterotoxin A gene (entA). Lysogeny results in loss of expression of the chromosomal beta-haemolysin gene (hlb) (negative conversion), the expression of staphylokinase and enterotoxin A (positive conversion), and the acquisition of resistance to lysis by all 23 phages of the International Basic Set (IBS) of S. aureus typing phages. Until this study, the basis of Phi42 resistance to lysis by exogenous phages was unknown. The authors report here that phage Phi42 encodes a restriction-modification (R-M) system, termed Sau42I, adjacent to and in the same orientation to the phage integrase gene int. The genes encoding Sau42I were cloned and sequenced, and found to consist of two overlapping reading frames, ORF S (specificity) and ORF RM (restriction-modification), in the same orientation. The ORFs share a high degree of DNA and amino acid sequence homology with the previously characterized BcgI R-M system of Bacillus coagulans. Expression of the cloned Sau42I ORF S and ORF RM in S. aureus 80CR3 transformants from a plasmid vector conferred resistance to lysis by all 23 IBS phages. Similarly, transformants of S. aureus RN4220 harbouring recombinant plasmids containing both ORFs were resistant to lysis by the IBS typing phages. However, transformants harbouring plasmids encoding either ORF S or ORF RM were susceptible to lysis by the IBS phages, and they had the same phage-susceptibility pattern as the respective parental isolates. In vitro analysis of crude and partially purified extracts of S. aureus transformants harbouring both the Phi42 ORF S and ORF RM genes indicated that Sau42I has endonuclease activity and requires co-factors Mg(2+) and S-adenosylmethionine in order to function, and activity is optimized at pH 8, although the precise recognition sequence has yet to be determined. The findings of this study confirm that Phi42 is a quadruple-converting phage, believed to be the first described for S. aureus, and show that it encodes a novel R-M system termed Sau42I.

Amino Acid Sequence↗
PubMed2004

Helicase-dependent isothermal DNA amplification.

Polymerase chain reaction is the most widely used method for in vitro DNA amplification. However, it requires thermocycling to separate two DNA strands. In vivo, DNA is replicated by DNA polymerases with various accessory proteins, including a DNA helicase that acts to separate duplex DNA. We have devised a new in vitro isothermal DNA amplification method by mimicking this in vivo mechanism. Helicase-dependent amplification (HDA) utilizes a DNA helicase to generate single-stranded templates for primer hybridization and subsequent primer extension by a DNA polymerase. HDA does not require thermocycling. In addition, it offers several advantages over other isothermal DNA amplification methods by having a simple reaction scheme and being a true isothermal reaction that can be performed at one temperature for the entire process. These properties offer a great potential for the development of simple portable DNA diagnostic devices to be used in the field and at the point-of-care.

Animals↗