Methods of nonspecific target capture of nucleic acids
a nucleic acid and target capture technology, applied in the field of molecular biology, can solve the problems of long time required to complete the nucleic acid isolation, complicated procedures, and add complexity to the design, optimization and performance of methods
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example 1
Nonspecific Target Capture of RNA
[0045]This example demonstrates the efficiency of various nonspecific target capture probe to capture RNA. Tests were performed by using a known amount of Chlamydia trachomatis rRNA (a mixture of 16S and 23S rRNA) which was hybridized to a labeled detection probe complementary to a sequence in the rRNA to label the target rRNA before it was captured. Typically, to prepare the test samples, 200 fmole of rRNA was hybridized with 1 pmole of a AE-labeled oligonucleotide in hybridization reagent (60 C for 60 min in 0.04 ml), and then cooled to room temperature (RT) and diluted with 0.3 ml of hybridization reagent. An aliquot (10 μl) of the probe-labeled target nucleic acid mixture was mixed with 0.5 ml of a substantially aqueous solution (made up of 0.2 ml sample transport reagent, 0.2 ml water and, 0.1 ml of TCR containing 50 μg of magnetic particles with poly-dT immobilized probes) and 20 pmoles of the nonspecific capture probe. The mixture was incubate...
example 2
Nonspecific Target Capture of RNA
[0055]This example demonstrates efficiencies of nonspecific target capture of rRNA by using a variety of different capture probes in assays performed substantially as described in Example 1 except that the target capture incubations were at RT for 10 to 30 min. All of the nonspecific capture probes used were synthesized using RNA bases and 2′-methoxy linkages and a 3′ DNA tail sequence.
[0056]In a first set of tests, the C9-containing nonspecific capture probes of SEQ ID Nos. 5 and 6 were tested using incubation at RT for 30 min, and the efficiency of RNA capture was 71% for capture probe of SEQ ID NO:5 and 89.5% for the capture probe of SEQ ID NO:6, compared to the positive control of 53% capture and the negative control of 1.3% capture. These results shown that incubation at 60 C was not needed for efficient nonspecific target capture of RNA.
[0057]In a second set of target captures, also incubated at RT for 30 min, the nonspecific capture probe of S...
example 3
Target Capture of HIV-1 Target RNA
[0062]This example demonstrates the use of two different nonspecific target capture probes to capture HIV-1 sequences, which were synthetic RNA sequences corresponding to portions of the protease-encoding gene of and the RT4 gene of HIV-1. Tests were performed individually for both target nucleic acids by using a known amount of in vitro RNA transcripts prepared from cloned Protease and RT4 sequences (a 681 nt Protease transcript, and a 471 nt RT4 transcript) which were hybridized specifically and individually to a labeled detection probe complementary to a sequence in the target rRNA before it was captured. To prepare the test samples, 200 fmole of the target RNA was hybridized specifically with 1 pmole of a AE-labeled detection probe oligonucleotide in hybridization reagent (60 C for 60 min in 0.04 ml), cooled to room temperature (RT) and diluted with 0.3 ml of hybridization reagent. An aliquot (10 μl) of the probe-labeled target RNA mixture was m...
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