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GSJ:Received May 13, 2005:
http://wbabin.net/saba/saba37.htm
Alternatives to Southern Hybridization and in situ Hybridization
James Saba
The previously described isothermal nucleic acid amplifications resulting in a spatially-localized product (1-4) would be excellent alternative means of Southern hybridization and in situ hybridization, as exemplified in the self explanatory Figure 1.
If desired non-latent primers could be used (5), however two hybridization steps would be needed. Of course, different labeling schemes such as beacons could be utilized.
In regards to utilizing restriction enzymes, if the 3' common sequence is sufficient, each thereof can prime a common isothermal amplification template.
When the 3' sequence is not sufficient in length, one could utilize several different template sequences, or alternatively a common universal nucleotide-containing common template as shown in Figure 2. Therein universal nucleotides are positioned adjacent to the sequence complementary to the 3' overhang.
This invention, as all the inventions described in this journal, is at the conceptual stage and a patent is anticipated. However, it is hoped that those with laboratories will investigate its full potential.
References:
2)
Spatially Localized Exponential Rolling Circle
Amplification (RCA)
Saba, JA Gen Sci J 2005 April 9-10
3)
Exceedingly Simple, Target Sequence-Stablized,
Latent Primers
Saba, JA Gen Sci J 2005 April 16
4)
Real-time Polymer Labeling via Incorporation of
FRET Dye-conjugated Monomers Adjacent to One Another
Saba, JA Gen Sci J 2005 April 18
5)
Poly-primed amplification of nucleic acid sequences
Kingsmore, et al United States Patent Application
20010041340
6)
Detection, Characterization and/or Quantitation of
Support-Affixed Nucleic Acid Sequences via Rolling
Circle Amplification (RCA).
Saba, JA 2005 April 15
7)
An alternative approach to deoxyoligonucleotides as
hybridization probes by insertion of deoxyinosine at
ambiguous codon positions
Ohtsuka E, et al J Biol
Chem. 1985 Mar 10;260(5):2605-8
8)
The applications of universal DNA base analogues
(review) Loakes, D Nucleic Acids Res. 2001 June 15;
29(12): 2437-2447
9)
Universal bases for hybridization, replication and
chain termination
Berger, et al Nucleic Acids Res.
2000 Aug 1;28(15):2911-4
10)
Hybridization properties and enzymatic replication
of oligonucleotides containing the photocleavable
7-nitroindole base analog.
Crey-Desbiolles, et al
Nucleic Acids Res. 2005 Mar 14;33(5):1532-43.
11)
Facile polymerization of dNTPs bearing unnatural
base analogues by DNA polymerase alpha and Klenow
fragment (DNA polymerase I).
Chiaramonte, et al
Biochemistry. 2003 Sep 9;42(35):10472-81
12)
Template directed incorporation of nucleotide
mixtures using azole-nucleobase analogs
Hoops, et al. Nucleic Acids Res. 1997 December 15; 25(24):
4866-4871.
13)
The Gly-952 residue of Saccharomyces cerevisiae
DNA polymerase alpha is important in discriminating
correct deoxyribonucleotides from incorrect ones
Limsirichaikul, et al J Biol Chem. 2003 May
23;278(21):19079-86. Epub 2003 Mar 7