Nucleic acid hybridization assays employing dA-tailed capture probes. II. Advanced multiple capture methods

Journal Article · Fri Sep 01 00:00:00 EDT 1989 · Analytical Biochemistry; (USA)

DOI:https://doi.org/10.1016/0003-2697(89)90256-X· OSTI ID:5102318

Hunsaker, W R; Badri, H; Lombardo, M; Collins, M L ^[1]

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1. Gene-Trak Systems, Framingham, MA (USA)

A fourth capture is added to the reversible target capture procedure. This results in an improved radioisotopic detection limit of 7.3 x 10(-21) mol of target. In addition, the standard triple capture method is converted into a nonradioactive format with a detection limit of under 1 amol of target. The principal advantage of nonradioactive detection is that the entire assay can be performed in about 1 h. Nucleic acids are released from cells in the presence of the (capture probe) which contains a 3'-poly(dA) sequence and the (labeled probe) which contains a detectable nonradioactive moiety such as biotin. After a brief hybridization in solution, the target is captured on oligo(dT) magnetic particles. The target is further purified from sample impurities and excess labeled probe by recapture either once or twice more on fresh magnetic particles. The highly purified target is then concentrated to 200 nl by recapture onto a poly(dT) nitrocellulose filter and rapidly detected with streptavidin-alkaline phosphatase using bromochloroindolyl phosphate and nitroblue tetrazolium. Using this procedure, as little as 0.25 amol of a target plasmid has been detected nonradioactively in crude samples in just 1 h without prior purification of the DNA and RNA. Finally, a new procedure called background capture is introduced to complement the background-reducing power of RTC.

• OSTI ID: 5102318
• Journal Information: Analytical Biochemistry; (USA), Vol. 181:2; ISSN 0003-2697
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
DNA
BIOASSAY
BIOTIN
ESCHERICHIA COLI
HYBRIDIZATION
OLIGONUCLEOTIDES
PHOSPHORUS ISOTOPES
PLASMIDS
RNA
TRACER TECHNIQUES
AZOLES
BACTERIA
CARBOXYLIC ACIDS
CELL CONSTITUENTS
HETEROCYCLIC ACIDS
HETEROCYCLIC COMPOUNDS
IMIDAZOLES
ISOTOPE APPLICATIONS
ISOTOPES
MICROORGANISMS
NUCLEIC ACIDS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANIC SULFUR COMPOUNDS
VITAMIN B GROUP
VITAMINS
550201* - Biochemistry- Tracer Techniques