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MOLECULAR BEACON PROBES

MIDLAND is licensed by The Public Health Research Institute of New York, Inc. to manufacture and sell Molecular Beacon Probes. These probes, first described by Dr. Fred Russell Kramer and his colleagues, make possible the in situ visual detection of target DNA sequences, and they also enable allelic discrimination and real-time PCR quantitation. Use of different fluorophores coupled with careful design of the probes permits distinguishing between sequences differing by as little as one base.

In the MIDLAND manufacturing and quality control process, the target sequence is also synthesized and the finished molecular beacon probe is tested using the synthetic target. The ratio of signal fluorescence (in presence of target) to background fluorescence (no target present) is measured and reported as analytical data accompanying the product. A copy of the UV/visible absorption spectrum of the purified molecular beacon probe also accompanies the product, as does a sample of the synthetic target sequence.

MOLECULAR PROBE BEACON PROBE PRICES
With 5'- 6-FAM, HEX, or TET with 3'- Dabcyl:
0.2 micromole scale synthesis:
(approximately 10-20 nanomoles purified molecular beacon probe)
$2.60/base of DNA + $325
With 5'- 6-FAM, HEX, or TET with 3'-Dabcyl:
1.0 micromole scale synthesis:
(approximately 50-100 nanomoles purified molecular beacon probe)
$3.30/base of DNA + $390
With 5'-Coumarin or Eosin with 3'-Dabcyl:
1.0 micromole scale synthesis:
(approximately 50-100 nanomoles purified molecular beacon probe)
$3.30/base of DNA + $575
0.2 micromole scale synthesis:
(approximately 10-20 nanomoles purified molecular beacon probe)
$2.60/base of DNA + $500
With 5'-Texas Red or Tetramethylrhodamine with 3'-Black Hole Quencher 2**:
1.0 micromole scale synthesis:
(approximately 50-100 nanomoles purified molecular beacon probe)
$3.30/base of DNA + $700
0.2 micromole scale synthesis:
(approximately 10-20 nanomoles purified molecular beacon probe)
$2.60/base of DNA + $600

* Under licensing agreements, any purchase of a Molecular Beacon Probe requires the purchase of a complementary target sequence available on a 50 nanomole scale of synthesis at (current price/base) of DNA.
MIDLAND will test and document the signal-to-noise ratio of the pair.
** Black Hole Quencher is a registered trademark of BioSearch Technologies, Inc.


Ordering Molecular Beacon Probes
Because of all the complexities associated with a molecular beacon including a variety of fluorophore and quencher combinations, please email your order or request to Dr. J. Lynn Myers at lmyers@oligos.com. There are a few guidelines in designing a successful Molecular Beacon. In brief, an oligonucleotide sequence is chosen that is complementary to the target sequence of interest. For real-time PCR, choose a target sequence in the middle of the expected amplicon. This sequence can be 10-40 nucleotides in length and should be free of secondary structure. A stem is formed by adding 5 -7 nucleotides at the 5'-end and adding 5-7 complementing nucleotides at the 3'-end. The structure is completed by appending a fluorophore at the 5'-end and a quencher at the 3'-end.

Excellent recommendations on the design of Molecular Beacon Probes can be found at www.molecular-beacons.com.

Beacon Designer software can also be used to design specific and efficient molecular beacons.

Molecular Beacon Probes
Molecular Beacon Probes are DNA oligonucleotides that become fluorescent when they hybridize to their target. They are hairpin-shaped, single-stranded molecules consisting of a probe sequence embedded between complementary sequences that form a hairpin stem. A fluorophore is covalently attached to one end of the oligonucleotide and a non-fluorescent quencher is covalently attached to the other end. In the absence of a target, the fluorophore is held close to the quencher and fluorescence cannot occur. When the probe binds to its target, the greater stability of the probe-target helix forces the stem to unwind, resulting in a separation of the fluorophore from the quencher, and fluorescence can occur. 1-5

Features of Molecular Beacon Probes include:
 
  • The ability to analyze several different targets in the same solution using differently-colored fluorophores
  • The ability to detect single-base mismatches and
  • Quantitation of nucleic acids
  •  
  1 Tyagi, S. and Kramer, F.R. (1996). Molecular beacons: probes that fluoresce upon hybridization. Nature Biotechnology 14:303-308.
  2 Kostrikis, L.G., Tyagi, S., Mhlanga, M.M., Ho, D.D., and Kramer, F.R. (1998). Spectral genotyping of human alleles. Science 279:1228-1229.
  3 Tyagi, S., Bratu, D.P., and Kramer, F.R. (1998). Multicolor molecular beacons for allele discrimination. Nature Biotechnology 16:49-53.
  4 Matsuo, T. (1998). In situ visualization of messenger RNA for basic fibroblast growth factor in living cells. Biochim. Biophys. Acta. 1379: 178-184.
  5 Piatek, A.S., Tyagi, S., Pol, A.C., Telenti, A., Miller, L.P., Kramer, F.R., and Alland, D. (1998). Molecular beacon sequence analysis for detecting
     drug resistance in Mycobacterium tuberculosis. Nature Biotechnology 16:359-363.


Molecular Beacons Probes are distinct from the Beacon technology, which is a registered trademark of Pan Vera Corporation, Madison, WI.

References to relevant Web sites
www.molecular-beacons.com/default.htm
www.stratagene.com/q_pcr/appnotes9.pdf

Guidelines for Choosing Oligonucleotide Sequences for Molecular Beacon Probes

In brief, an oligonucleotide sequence is chosen that is complementary to the target sequence of interest. For real-time PCR, choose a target sequence in the middle of the expected amplicon. This sequence can be 10-40 nucleotides in length and should be free of secondary structure. A stem is formed by adding 5 -7 nucleotides at the 5'-end and adding 5-7 complementing nucleotides at the 3'-end. The structure is completed by appending a fluorophore at the 5'-end and a quencher at the 3'-end.

FLUOROPHORE/QUENCHER COMBINATIONS (*)

Recommended

  Maximum (nm)
Quencher Fluorophore Excitation Emission
Dabcyl    Coumarin 434 475
Dabcyl    6-Fam (Fluorescein) 494 521
Dabcyl    TET 519 537
Dabcyl    Eosin 524 544
Dabcyl    HEX 535 556
Black-Hole 2    Tetramethylrhodamine 558 580
Black-Hole 2    Texas Red 592 615
* Please call or e-mail us if the desired fluorophore is not listed.

The 5' nuclease detection assay and other homogeneous amplification methods used in connection with Polymerase Chain Reaction ("PCR") process are covered by patents owned by Roche Molecular Systems, Inc. and F. Hoffmann-La Roche Ltd ("Roche"). The use of these methods requires a license. No license under these patents (including, but not limited to, United States Patent Nos. 5,210,015; 5,487,972; 5,804,375; and 5,994,076) to use the 5' nuclease assay or any Roche patented homogeneous amplification process is conveyed, either expressly or implied, to the purchaser of any MIDLAND product. Purchasers of these products must obtain a license to use the 5' nuclease or homogeneous PCR process before performing PCR. Further information concerning licenses to practice the PCR process may be obtained by contacting:
The Licensing Specialist
Roche Molecular Systems, Inc.
1145 Atlantic Avenue
Alameda, California 94501
(510) 814-2984

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