DNA chip, PNA chip, and their preparation methods

Details DNA chip, PNA chip, and their preparation methods DNA chip, PNA chip, and their preparation methods

2000-06-07

2004-03-30

Siew, Jeffrey (Department: 1637)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S007100, C435S091100, C435S091200, C435S287100, C536S022100, C536S023100, C536S024300, C536S024310, C536S024320, C536S024330

Reexamination Certificate

active

06713255

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to a DNA chip or a PNA chip favorably employable for detecting, with high sensitivity, a nucleic acid fragment complementary to a DNA fragment or a PNA fragment of the DNA chip or the PNA chip, respectively. The invention further relates to a method of quantitative analysis of a nucleic acid fragment contained in a liquid sample in an extremely small amount using a DNA chip or a PNA chip which has a DNA fragment or a PNA fragment, respectively, on its solid carrier.
BACKGROUND OF THE INVENTION
Detection of a nucleic acid fragment is generally made using a probe DNA which is complementary to the nucleic acid fragment to be detected, by way of hybridization. The probe DNA is generally fixed onto a solid carrier (substrate) to give a DNA chip. In more detail, a nucleic acid fragment in a sample liquid is once labelled with a fluorescent label or a radioisotope label, and then the sample liquid is brought into contact with the probe DNA of the DNA chip If the labelled nucleic acid fragment in the sample liquid is complementary to the probe DNA, the labelled nucleic acid fragment is combined with the probe DNA by hybridization. The labelled nucleic acid fragment fixed onto the DNA chip by hybridization with the probe DNA is then detected by an appropriate detection method such as fluorometry or autoradiography. The DNA chip is widely employed in gene technology, for instance, for detecting a complementary nucleic acid fragment or sequencing a nucleic acid.
The DNA chip is described, for instance, in Fodor S. P. A., Science, 251, 767(1991) and Schena M., Science, 270, 467(1995). The DNA chip is understood to efficiently detect a small amount of a complementary nucleic acid fragment in a small amount of a sample liquid. However, the heretofore known DNA chip has a limitation on the detection level of nucleic acid fragment such as a level of not less than 10
−19
mol./dot. in certain cases, an improved high sensitive detection is required. For instance, a procedure for monitoring of gene expression, particularly gene expression of a low level, a procedure for analyzing a gene variation, and a procedure for analyzing gene polymorphism, require higher sensitivity in the detection of complementary nucleic acid fragments.
Detection of nucleic acid fragment using an electrochemical label is also known (Japanese Patent Provisional Publication No. 9-288080, and a preprint of the 57th Analytical Chemistry Conference pp. 137-138 (1996)). The electrochemical label such as N-hydroxysuccinimide ester of ferrocenecarboxylic acid is attached to a probe DNA. The probe DNA is fixed onto an electroconductive substrate having an output terminal. In the detection procedure, a sample liquid containing the target nucleic acid fragment is brought into contact with the probe DNA having the ferrocene derivative label in the presence of an electrochemically active thread intercalator. The target nucleic acid fragment, if it is complementary to the probe DNA, is hybridized with the probe DNA. Into the formed hybrid structure, the electrochemically active thread intercalator is intercalated. Thereafter, a potential is applied to the electroconductive substrate to measure an electric current flowing through the ferrocene derivative label and the thread intercalator.
On the above-mentioned electroconductive substrate are fixed the ferrocene derivative-modified DNA fragments of approximately 10
−11
mol., per 1 mm
2
of surface area of the substrate). It is described that complementary nucleic acid molecules in the range of 10
−15
to 10
−11
/mm
2
are detected, when a sample liquid containing target nucleic acid molecules is brought into contact with the probe DNA.
Preprint of the 47th Polymer Society Conference, pp. 3155-3156 (1998) describes an electrochemical detection method in which a sample liquid containing a target nucleic acid fragment is brought into contact a DNA probe fixed onto an electroconductive substrate in the presence of an electrochemically active thread intercalator. On the electroconductive substrate are fixed the DNA probes in an amount of approximately 10
−11
mol./2 mm
2
(surface area of substrate). If a sample liquid containing target nucleic acid fragments in an excessive amount (such as approximately ten times or more) is brought into contact with the probe DNA, complementary nucleic acid fragments in an amount of approximately 10
−11
mol. are detected.
P. E. Nielsen et al., Science, 254, 1497-1500(1991) and P. E. Nielsen et al., Biochemistry, 36, pp.5072-5077 (1997) describe PNA (Peptide Nucleic Acid or Polyamide Nucleic Acid) which has no negative charge and functions in the same manner as DNA does. PNA has a polyamide skeleton of N-(2-aminoethyl)glycine units and has neither glucose units nor phosphate groups. A representative PNA as well as a representative DNA are illustrated below:
Since PNA is electrically neutral and is not charged in the absence of an electrolytic salt, PNA is able to hybridize with a complementary nucleic acid fragment to form a hybrid which is more stable than hybrid given by a DNA prove and its complementary nucleic acid fragment (Preprint of the 74th Spring Conference of Japan Chemical Society, pp. 1287, reported by Naomi Sugimoto).
Japanese Patent Provisional Publication No. 11-332595 describes a PNA probe fixed on a solid carrier at its one end and a detection method utilizing the PNA probe The PNA probe is fixed onto the solid carrier by the avidin-biotin method.
The aforementioned P. E. Nielsen et al., Science, 254, 1497-1500(1991) also describes a PNA probe labelled with isotope element and a detection method of a complementary nucleic acid fragment.
Since the PNA probe shows no electric repulsion to a target nucleic acid fragment in a sample liquid, an improved high detection sensitivity is expected.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide a nucleic acid fragment-detecting means showing an improved high sensitivity for a nucleic acid fragment contained in a sample liquid.
Specifically, it is an object of the invention to provide a DNA chip and a PNA chip which are employable for quantitatively detecting a nucleic acid fragment contained in a sample liquid in an extremely small amount.
It is another object of the invention to provide a method for quantitatively detecting, with a high sensitivity and good reproducibility, a nucleic acid fragment contained in a sample liquid in an extremely small amount.
The present invention resides in a DNA chip comprising a solid carrier and a plurality of DNA fragments fixed onto the solid carrier at each one end, wherein a plurality of short chain spacer molecules having a hydrophilic moiety at each one end are fixed at each another end onto a surface area of the solid carrier having no DNA fragments thereon.
The invention further resides in a PNA chip comprising a solid carrier and a plurality of PNA fragments fixed onto the solid carrier at each one end, wherein a plurality of short chain spacer molecules having a hydrophilic moiety at each one end are fixed at each another end onto a surface area of the solid carrier having no PNA fragments thereon.
The DNA chip (or PNA chip) of the invention is favorably prepared by a process comprising the steps of:
applying onto a solid carrier an aqueous solution of a plurality of DNA fragments (or PNA fragments) dissolved or dispersed in an aqueous medium to fix the DNA fragments (or PNA fragments) onto the solid carrier; and
applying onto the solid carrier having thereon the fixed DNA fragments (or PNA fragments) an aqueous solution of short chain spacer molecules having at each one end a hydrophilic moiety and at each another end a moiety reactive to fix to the solid carrier.
In the DNA chip and PNA chip of the invention, the solid carrier preferably is an electro-conductive substrate.
The DNA chip (or PNA chip) of the invention is favorably employed in a method of quantitative analysis of a nucleic acid fragment contained in a sample liquid which is

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