Separate production of single-stranded DNA is not necessary: circuit denaturation of double-stranded DNA followed by hybridization of single strands on oligonucleotide microchips.
An approach to circuit renaturation-hybridization of dsDNA on oligonucleotide microchips is described. A close circuit cycling device has been developed, and the feasibility of the proposed technique was demonstrated on two platforms. First, a commercial microchip for detection of rifampicin resistance in Mycobacterium tuberculosis was used. Hybridization of a 126 nt long single-stranded DNA (ssDNA) fragment of the rpoB gene according to manufacturer's protocol has been compared to hybridization of the same double-stranded DNA (dsDNA) fragment using the developed approach. Hybridization signals obtained by both methods were comparable in intensity and correlated closely. Second, a 22 nt long hairpin-forming oligonucleotide was designed and hybridized with a custom microchip containing probes complementary to both strands of the oligonucleotide. Conventional hybridization of this oligonucleotide did not yield any significant signals. Cleavage of the hairpin loop resulted in the formation of a 9 bp long intermolecular duplex. Hybridization of the duplex using the suggested technique yielded strong signals. The proposed approach allows analyzing target DNA in double-stranded form bypassing the preparation of single-stranded targets. Moreover, both complementary chains could be analyzed simultaneously, providing a reliable internal control. Being combined with fragmentation this method opens new possibilities in analyzing ssDNA with complex secondary structure.
Vasiliskov
Vadim A
VA
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov str. 32, Moscow, Russia. vadimav@biochip.ru
Chudinov
Alexander V
AV
Chechetkin
Vladimir R
VR
Surzhikov
Sergey A
SA
Zasedatelev
Alexander S
AS
Mikhailovich
Vladimir M
VM
D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D004277:DNA, Single-Stranded / Q000737:chemistry*; D012321:DNA-Directed RNA Polymerases; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D009841:Oligonucleotides / Q000737:chemistry*
D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D009841:Oligonucleotides; C505805:rpoB protein, Mycobacterium tuberculosis; D004247:DNA; D012321:DNA-Directed RNA Polymerases