Data processing: measuring – calibrating – or testing – Measurement system in a specific environment – Biological or biochemical
Reexamination Certificate
2001-02-20
2004-03-16
Brusca, John S. (Department: 1631)
Data processing: measuring, calibrating, or testing
Measurement system in a specific environment
Biological or biochemical
Reexamination Certificate
active
06708119
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to an automatic vector unit removing method for automatically removing a part of the vector inside a fragment of an object DNA when it is taken out of a proliferated vector in a DNA cloning. The DNA cloning process is performed to proliferate a fragment of the object DNA by chemically bonding a clone, that is, a fragment of a DNA containing a gene to be proliferated to a DNA molecule called a vector, and then proliferating the vector in cells such as
Escherichia coli,
etc.
2. Description of the Related Art
A nucleic acid is formed by nucleotide composed of a base, pentose, and phosphoric acid. The nucleotide is a compound of a nucleoside and a phosphoric acid. The phosphoric acid forms a polymer through the nucleoside to produce either a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).
The bases forming part of the nucleic acid can be a purine or a pyrimidine. The purine can be an adenine A or a guanine G while the pyrimidine can be a cytosine C or a thymine T.
The DNA having the composition called a polynucleotide strand is formed by a strand of the above listed four bases, that is, the adenine A, guanine G. cytosine C, and thymine T, bound in a series. For example, if a DNA is extracted from the chromosome in the cell of a human being and is arranged as a sequence, it can be as long as 1 meter and contain 3 billion bases.
Thus, a DNA has a strand of bases, that is, a base sequence linked in the form of a strand. The strand is normally very long. In genetic engineering, a DNA including venous genes is cleaved for gene recombination, and a DNA fragment having specific genetic information is extracted from a number of the cleaved DNA fragments. The extracted DNA fragments, that is, object DNA fragments, should be normally proliferated.
Normally, a unique sequence of object DNA fragments is very small in volume, and the object DNA fragments are combined with a vector to perform the cloning for DNA sequencing.
To attain this, the object DNA fragments are chemically bound to the DNA called a vector which is normally a circular DNA. The combination of the object DNA fragment and the vector DNA, that is, a recombinant DNA, is integrated into an appropriate cell such as a colibacilli (colon bacillus), and the cell is proliferated to produce a large volume of the recombinant DNA. As a result, the cloning process of generating a large volume of the object DNA fragments is successfully performed.
A vector is commonly a DNA having a double helix structure a specific portion of which is cleaved using some restriction enzymes. The object DNA fragment is integrated into the cleaved portion. Before describing the cleaving of the DNA using the restriction enzyme, the structure of the DNA is explained below.
A DNA has the structure of a base sequence, that is, a sequence of bases bound in the form of a strand. Since the DNA strand is directional, ATGCACGA→ is different from ATGCACGA← (which equals AGCACGTA→).
Both ends of the DNA strand are named. Accordingly, the end provided with a hydroxyl group at the position of 3′ of a saccharum is called a 3′ end. The other end, that is, the end provided with a phosphate group at the position of 5′ of a saccharum is called a 5′ end. When the DNA strand is described, the 5′ end is positioned on the left while the 3′ end is positioned on the right.
A DNA normally exists in a double-stranded state as two complementary and anisotropic base sequences. In the two base sequences, the facing bases have a fixed relationship, and the adenine A faces the thymine T, while the guanine G faces the cytosine C. An example of a DNA double-strand is shown as follows:
5′end ATGCATGCTAGCTAGCT 3′end (strand A) (SEQUENCE ID NO. 1)
|||||||||||||||||
3′end TACGTACGATCGATCGA 5′end (strand B) (SEQUENCE ID NO. 2)
Strand B is complementary to strand A and is represented as a single strand as follows:
5′end AGCTAGCTAGCATGCAT\ 3′end (strand B) (SEQUENCE ID NO. 3).
Thus, the DNA represents a genetic meaning with the two complementary base sequences as a pair. The base sequences unique to the restriction enzyme are identified to shear the DNA at the identified points.
FIG. 1
shows how the DNA base sequence is cleaved using the restriction enzyme. In
FIG. 1
, the restriction enzyme called HpaI shears the DNA at the same positions on the two strands of the DNA whereas the restriction enzymes EcoRI and Hind III shear double strands at different points on the two strands.
As shown in
FIG. 1
, a number of restriction enzymes can identify nucleotide sequences formed by 6 pairs of bases. The two nucleotide strands in the identification area, that is, the site of the restriction enzyme, are arranged in the opposite directions. Most restriction enzymes indicate different cleaving positions on two strands, thereby forming uneven ends, that is, cohesive ends. The above described object DNA fragments are integrated into the positions where the DNA is cleaved based on the restriction enzymes.
FIG. 2
shows how to mount the object DNA fragments in the vector. In
FIG. 2
, the circular plasmid DNA molecule is cleaved by the restriction enzymes to obtain linear plasmid DNA molecules having cohesive ends. A plasmid is contained in, for example, bacteria, and can autonomously proliferate unlike the chromosome DNA. The linear plasmid DNA molecule and the object DNA fragments, that is, one of various DNA fragments obtained by cleaving the chromosome DNA using the restriction enzymes, form base pairs. This is referred to as annealing at cohesive ends, thereby forming a circular DNA.
Thus, the cohesive ends generated by the restriction enzymes are required for the recombinant DNA technology. Actually, any DNA fragments can be bound to a plasmid DNA by cleaving the DNA using the restriction enzymes used in generating the object DNA fragments. The linear plasmid DNA molecule is bound to the object DNA fragments through the DNA ligase for repair of the cleaved portion in a single strand of the double stranded DNA, thereby generating a chromosome-DNA-integrated plasmid DNA molecule.
The generated plasmid DNA molecule can be proliferated in bacteria or enzymes. The process is called a DNA cloning technology.
FIG. 3
shows the vector used in the DNA cloning process and the multiple cloning site in the vector. A number of restriction enzyme sites to be cleaved by various restriction enzymes is concentrated in the multiple cloning site.
When an object DNA fragment is taken out of a large amount of the plasmid DNA molecules generated as a result of the DNA cloning process, the nucleotide sequence of the DNA fragment processed in the cloning operation should be correctly determined and the bases in unnecessary portions are deleted to take out a DNA fragment having a correct structure. To determine the nucleotide sequence in the DNA fragment, a DNA sequencer is used to automatically read the DNA base sequence.
To know the sequence of A, G, T, and C in the DNA is to understand the genetic information. The sequence technology for determining the base sequence has advanced with the technologies of other fields, and is closely related to the discovery of the restriction enzymes and nucleic acids, and the development in technology for DNA cloning, nucleic acid chemistry, etc.
Recently, computer technology has been utilized as one of the sequence methods, thereby enabling an enormous volume of data to be input end accumulated. Thus, computers are required in determining the base sequence.
With the
Imai Kensaku
Kitajima Masato
Brusca John S.
Fujitsu Limited
Staas & Halsey , LLP
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