Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2002-03-21
2004-10-26
Horlick, Kenneth R. (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S019000, C435S023000
Reexamination Certificate
active
06808885
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention is generally directed to a triple-stranded DNA, a method of forming the same, and Southern hybridization employing the same.
2. Discussion of the Background
Conventional methods are known to form a complex including double-stranded and single-stranded DNAs. That is to say, as shown in
FIG. 19
, a target DNA (i.e. a double-stranded DNA) and a probe DNA (i.e. a single-stranded DNA) are prepared. The probe DNA (i.e. the single-stranded DNA) has a base sequence which is substantially complementary to a portion of a base sequence of one of DNA-chains of the target DNA. These DNAs and a RecA of
Escherichia coli
are mixed into a solution which includes a buffer etc and the resultant mixture is held at a predetermined temperature for a sufficient time duration.
Then, a DNA-protein complex is obtained which is made up of the target DNA, the probe DNA, and the RecA protein. In detail, first of all, the RecA protein is bound to the probe DNA to form a probe DNA-RecA protein complex. Subsequently, the resultant complex or the probe DNA-RecA protein complex is bound to the target DNA to form a DNA-protein complex which includes a three-chain formation region. At this stage, the probe DNA is believed to bind to a region of the target DNA which has a base sequence complementary to the probe DNA. The DNA-protein complex at this state, though it has the three-chain formation region, is relatively stable (see B. Jagadeeshwar Rao et al., Proc. Natl. Sci. USA, 88,2984-2988 (1991), Gurucharan Reddy at al., Biochemistry, 33,11486-11492(1994), and Efim I. Golumb eat al., Mutation Research, 351, 117-124 (1996)).
However, as shown in
FIG. 19
, if the RecA protein is deactivated in such a manner that the DNA-protein complex is mixed with a sodium dodecyl sulfate (SDS) and/or a protein splitting enzyme (e.g. protease K) and the resultant mix is held at a temperature for a sufficient time duration, the bonding between the target DNA and the probe DNA dissociates in addition to a deleting the RecA protein from the DNA-protein complex. That is to say, the structure of the DNA-protein is stable by the presence of RecA protein and without RecA protein it is impossible to form or produce a triple-stranded DNA.
Therefore, in the field of biogenetics or the like, if one requires or wishes to use a triple-strained DNA, there are many restrictions as long as the triple-stranded DNA must remain in a complex with RecA Thus, a need exists to develop a method of forming a RecA protein free triple-stranded DNA whose structure remains stable.
SUMMARY OF THE INVENTION
Accordingly in order to meet the above need, the present invention provides a triple-stranded DNA whose structure can remain stable even if no protein is contained in its complex, a method of forming such a triple stranded DNA, and Southern hybridization employing such a triple-stranded DNA.
A first aspect of the present invention is to provide a method for forming a three-stranded DNA which comprises the steps of:
(a) DNA-protein complex forming process for forming a DNA-protein complex, wherein (1) a linearized double-stranded DNA, (2) a linearized single-stranded DNA including a base sequence, the base sequence being substantially complementary to a base sequence which extends from a base near 5′-end of one of DNA chains of the double-stranded DNAs, (3) a recombinant protein which is at least one of a homologous protein and another protein which is similar thereto in function, and (4) a nuclease which is at least one of an Exonuclease I of
Escherichia coli
and another protein which is similar thereto in function are reacted in order that in the DNA-protein complex an end neighboring inclusion region including the 5′-end of one of the DNA-chains of the double-stranded DNAs is bound to a complementary region including the substantially complementary base sequence of the single-stranded DNA under a participation of at least the recombinant protein; and
(b) protein deactivating process deactivating both the recombinant protein and nuclease to bind the complementary region of the single-stranded DNA to the end neighboring inclusion region of the double-stranded DNA.
In accordance with the first aspect of the present invention in the DNA-protein complex forming process, the DNA-protein complex is formed from the double-stranded DNA, the recombinant protein, and the nuclease. Thereafter, in the subsequent protein deactivating process, deactivating the recombinant protein and the nuclease makes it possible to form the triple-stranded DNA which has the 3-chain forming region which is formed by the bonding between the end neighboring inclusion region of the double-stranded DNA and the complementary region of the single-stranded DNA. Thus-formed triple-stranded DNA can remain its structure i.e. cannot be disassociated, even if a heat is applied thereto more or less, without having to pare a specially prepared protein such as RecA for stabilizing the structure. It is to be noted that the present invention makes it possible to form the 3-chain forming region on both of the end neighboring inclusion regions of the double-stranded DNA other than the formation on one of the end neighboring inclusion regions.
The above-mentioned method for forming a triple-stranded DNA is applicable to, say, southern hybridization.
In the conventional southern hybridization, the operations, for example, are as follows. As a target DNA a restriction-enzymatically cleaved linearized double-stranded DNA is prepared, while as a probe DNA a single-stranded DNA is prepared whose 5′-end is labeled with 32P with usage of T4 Polynucleotide kinase and [&ggr;-32P]. The target DNA (i.e. the double-stranded DNA) is subjected to agarose gel electrophoresis and the agarose gel is placed on a membrane for vacuum filtration or the like and the target DNA (i.e. the double-stranded DNA) in the agarose gel is transfer onto the membrane. Thereafter, the target DNA (i.e. the double-stranded DNA) is made into a single-stranded state by disassociation as well as the resulting target DNA (i.e. the double-stranded DNA) is made immobilized on the membrane. Then, the resulting membrane is immersed into a solution of the probe DNA (i.e. a solution of the labeled single-stranded DNA) for hybridization and the membrane is made cleaned. Thereafter, the membrane is taken with a picture of autoradiogram to record a signal on an X-ray film which results from the labeled probe, DNA (i.e., the labeled single-stranded DNA).
On the other hand, a southern hybridization which depends on the present invention can be performed, for example, according to the following steps. In detail, like the conventional southern hybridization, a target DNA (i.e. a double-stranded DNA) and a labeled probe DNA (i.e. a labeled single-stranded DNA) are prepared At this stage, if the present invention is employed to do southern hybridization, a DNA-protein complex is formed by reacting such DNAs, a recombinant protein, and a nuclease (DNA-protein complex forming process). Thereafter, the recombinant protein and the nuclease are made deactivated to form a stable triple-stranded DNA having a 3-chain forming region (Protein deactivating process). Next, the resulting triple-stranded DNA is subject to agarose gel electrophoresis. Thereafter, the agarose gel is placed on filter paper to dry with drying device. The resulting gel is taken with a picture of autoradiogram to record a signal on an X-ray film which results from the labeled probe DNA (i.e. the labeled single-stranded DNA).
Thus-performed southern hybridization according to the present invention can be of less time operation and less cumbersome, when compared to the conventional southern hybridization. The reason is that southern hybridization according to the present invention eliminates skilled and/or long-time required operations such as transfer of DNA in agarose gel on membrane, immersing such membrane into probe DNA solution, and membrane cleaning. It is to be noted that the abov
Aisin Seiki Kabushiki Kaisha
Horlick Kenneth R.
Wilder Cynthia
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