WO2002097083A1

WO2002097083A1 - Method of extending long-chain dna

Info

Publication number: WO2002097083A1
Application number: PCT/JP2001/007522
Authority: WO
Inventors: Noritada Kaji; Masanori Ueda; Yoshinobu Baba
Original assignee: Japan Science And Technology Corporation
Priority date: 2001-05-29
Filing date: 2001-08-31
Publication date: 2002-12-05
Also published as: JP2003018985A

Abstract

A method of extending a long-chain nucleic acid by using a gel and a low frequency AC electric field which makes it possible to conveniently and quickly collect and re-use an extended and analyzed long-chain DNA; nucleic acid molecules extended by this method; and a method of extending nucleic acid molecules involving the step of providing the nucleic acid molecules in a gel and the step of applying a low-frequency AC electric field to the nucleic acid molecules in the gel.

Description

Description Method for extending long-chain DNA

Technical field

The present invention relates to a method for extending a nucleic acid molecule. More specifically, the present invention relates to a method for extending a nucleic acid molecule using a gel and a low-frequency AC electric field. Background art

Sequencing of the human genome has ended much faster than originally planned. With the advance of such DNA analysis technology, the ultimate single-molecule genome analysis technology that reads the information from only one chromosomal DNA molecule will be important in the future. Technology for freely manipulating DNA with one molecule to realize single-molecule genome analysis is required. Traditional single DNA molecule manipulation methods have used optical tweezers, atomic force microscopy, and high-frequency AC electric fields, but these methods require pretreatment or manipulation of DNA. It has drawbacks such as difficulty in recovering DNA and using it as a new sample, and inability to extend long-chain DNA such as chromosomal DNA.

The total size of human chromosomal DNA is 3.5 billion salt pairs (3.5 gigabase pairs: Gbp), and the smallest chromosome 21 has 48 million base pairs (48 megabase pairs: Mbp). Have. Therefore, in the process of separating and identifying fragments obtained by cutting chromosomal DNA and reconstructing the original sequence, it is advantageous to treat the chromosomal DNA as it is, without breaking it into pieces. Therefore, a technique for rapidly and precisely analyzing a large fragment of the order of Mbp is desired. Disclosure of the invention

The present invention relates to a method for extending a long-chain nucleic acid using a gel and a low-frequency AC electric field, as a method for recovering and reusing the long-chain DNA analyzed by extension in a simple and rapid manner. It is an object to provide a nucleic acid molecule extended by the method. That is, the gist of the present invention is:

(1) a method for extending a nucleic acid molecule, comprising the steps of: placing a nucleic acid molecule in a gel; and applying a low-frequency AC electric field to the nucleic acid molecule in the gel.

(2) The method according to (1) above, wherein the length of the nucleic acid molecule is 23 to 570 Okbp.

(3) The method according to (1) or (2), wherein the frequency of the low-frequency AC electric field is 1 to 100 Hz.

(4) The method according to any one of (1) to (3), wherein the intensity of the low-frequency AC electric field is I 10 I to I 10000 I (absolute value) VZcm.

(5) The method according to any one of (1) to (3), wherein the intensity of the low-frequency AC electric field is gradually increased.

(6) The method according to (5), wherein the intensity of the low-frequency AC electric field is gradually increased from I 10 I VZ cm to I 300 I (absolute value) V cm.

(7) The method according to (5) or (6), wherein the rate of gradually increasing the intensity of the low-frequency AC electric field is I 1 I to I 100 I (absolute value) V / cm · s.

(8) The method according to (1) to (7), wherein the frequency of the low-frequency AC electric field is gradually increased.

(9) The method according to (8), wherein the frequency of the low-frequency AC electric field is gradually increased from 1 Hz to 20 Hz.

(10) The method according to (8) or (9), wherein the speed of gradually increasing the frequency of the low-frequency AC electric field is 0.01-1HZZS.

(11) The method according to (1), further comprising a step of detecting and recovering the extended nucleic acid molecule. (10) The method according to any of the above,

(12) a nucleic acid molecule stretched by the method according to any of (1) to (11),

About. BRIEF DESCRIPTION OF THE FIGURES

Fig. 1 is a schematic diagram of the experimental device. Two Pt electrodes were placed in a miniature cell at a distance of 1.00111 or 0.5 cm. The AC electric field generated by the arbitrary waveform generator was increased to an appropriate intensity by a high-speed power amplification bipolar power supply, and applied between Pt electrodes. The symbols in the figure represent the following:

1 High-speed power amplification bipolar power supply;

2 Arbitrary waveform generator;

3 100x oil immersion objective lens;

4 30 mm x 40 mm cover glass;

5 Pt electrode;

6 agarose gel;

7 0.5 XT BE buffer;

8 DNA samples.

FIG. 2 is a diagram showing the extension process of T 4 DNA in 1% agarose gel under an alternating electric field. Continuous fluorescence image of T4 DNA stained with YOYO 1. At 0 s, an AC electric field (200 VZcm, 10 Hz) was applied horizontally to T4DNA in a random coil-like state. B) Plot of the maximum diameter 1 ^ of T4DNA (indicated by A) after the application of an AC electric field.

FIG. 3 is a diagram showing a plot of the average maximum diameter 1 ^ with respect to the frequency f of the external electric field of T4DNA. The maximum diameter 1 ^ of each molecule was also time-averaged for 10 seconds (black circles) and about 7 seconds (open circles) for 1 to 5 different molecules at each frequency. The horizontal dashed line The figure shows the equilibrium value of T4 DNA in a random coil-like state in the absence of an external electric field.

4 is a diagram showing a time series of the average maximum diameter of 1 ^ and the center of gravity of the X component G _x of T4 DNA at 200 VZcm under different external frequency. The dark line indicates, and the light line indicates G _x . A) 1 Hzo B) 1 OHZo C) 10 OHz FIG. 5 is a diagram showing the average maximum diameter 1 ^ and the X component G _{x of the} center of gravity of T 4 DNA expanded at different electric field intensities. A) 1 5 0 V / cm . 50VZcm, and 25 0 VZcm plots of R! And G _x under electric field. B) Fluorescence images of T4 DNA under electric fields of 150 V / cm, 50 V / cm. And 250 VZcm. FIG. 6 is a diagram showing a plot of the average maximum diameter 1 ^ against the intensity of the external electric field of T 4 DNA. Dashed lines indicate the boundaries between stretched and unstretched regions.

FIG. 7 is a view to view the time series of the average maximum diameter R, and T4DNA of the center of gravity of the X component G _x. The dark line indicates, and the light line indicates G _x . A) 50 V / cm, 1 OHZo B) 200 V / cm, 10 Hz ₀ C) 600 V / cm, 10 Hz ₀ Figure 8 shows the average maximum diameter 1 ^ for the agarose gel concentration of T4 DNA. It is a figure showing a plot. The dashed line is in low molecular solution.

FIG. 9 shows the extension of Saccharomyces' Celepiche DNA having a length of about 285 kbp under an alternating electric field (200 V / cm, 1 OHz). A) Fluorescence image of expanded Saccharomyces cerevisiae chromosomal DNA. B) Fluorescence image trace. BEST MODE FOR CARRYING OUT THE INVENTION

The method for extending a nucleic acid molecule of the present invention includes a step of arranging a nucleic acid molecule in a gel and a step of applying a low-frequency AC electric field to the nucleic acid molecule in the gel. Here, the nucleic acid molecule is The term “stretch” means that a nucleic acid molecule having a higher-order structure (for example, a coil, a bent structure, or the like) is oriented parallel to the direction of an electric field to be in an extended state.

Examples of the nucleic acid molecule applied to the extension method of the present invention include double-stranded DNA, the length of which is preferably 23 to 5700 kbp, more preferably 48 to 2200 kbp. According to the method described above, the present invention can also be applied to long chains such as 2200 to 5570 kbp. The amount of the nucleic acid molecule applied to the extension method is preferably from 6.25 to 394 ngZml, more preferably from 20 to 394 ngZml, from the viewpoint of one-molecule observation.

The gel used in the nucleic acid extension method of the present invention may be any gel, such as agarose gel or acrylamide gel, as long as it can introduce nucleic acid molecules. The gel concentration to be used is preferably 0.5 to 3.0% by weight, more preferably 0.5 to 2.5% by weight, and particularly preferably 1.0 to 2.0% by weight from the viewpoint of maintaining the gel structure. . The size of the gel to be used is appropriately determined without any particular limitation. Generally, a gel of 1 to 100 mm X I to 100 mm x l to 100 mm is used.

In the step of applying a low-frequency AC electric field to the nucleic acid molecules in the gel, it is generally preferable to use a buffer, for example, TBE (tris-borate, EDTA), TB (tris-borate), TAE (tris-borate). -Acetate, EDTA), buffer solutions such as FI SH buffer SSC (sodium chloride monosodium chloride), denaturing buffer (formamido SSC, dextran sulfate-formamide SS) are used. For example, when the DNA to be sampled is a solution, a method for disposing the sample solution is to drop the sample solution onto a pre-made gel on a DNA extension gel and introduce the DNA solution into the DNA extension gel by a DC electric field. If the sample DNA is contained in the gel plug, prepare a DNA extension gel around the gel plug and introduce it into the DNA extension gel with a DC electric field. If that And the like.

In the present invention, the term “low-frequency AC electric field” means that when a voltage is applied in one direction to a positive direction such as a sine wave, a rectangular wave, or a triangular wave having a frequency of 1 to 10 OHz, the waveform has the same intensity as the waveform. Electric field. The low frequency alternating electric field can be generated, for example, by using a conventional frequency generator.

The frequency of the low-frequency AC electric field is preferably from 1 to 10 OHz, more preferably from 1 to 20 Hz, and particularly preferably from 5 to 15 Hz, from the viewpoint of drawing out the entanglement effect between the DNA and the gel and effectively driving the DNA. preferable.

The intensity of the low-frequency AC electric field is preferably from 1101 to 11000001 V / cm from the viewpoint of drawing out the entanglement effect between DNA and the gel and effectively driving DNA, and I10I to I1. 000 IV / cm is more preferable, I10I to I450IV / cm is more preferable, and I200I to I300Icm is particularly preferable. If the nucleic acid molecule to be extended is a long-chain DNA having a length exceeding 500 kbp, the intensity of the low-frequency AC electric field should be low to prevent irreversible entanglement between the DNA molecule and the gel fiber. It is more preferable to increase from.

In this case, it is preferable to gradually increase the intensity of the low-frequency AC electric field from 110001 V / cm to 13001 V / cm from the viewpoint of preventing irreversible entanglement between the DNA molecules and the gel fibers. It is more preferable to gradually increase from I 50 I cm to I 300 I VZ cm, particularly preferably from I 10 I VZ cm to I 300 I cm.

From the viewpoint of preventing irreversible entanglement between DNA molecules and gel fibers, the rate of gradually increasing the strength of the low-frequency AC electric field is preferably from I 1 I to I 100 I VZcm · s, and from 15 to IV / cm · s is more preferable, and 110 to 1101 VZ em's is particularly preferable.

When the nucleic acid molecule to be extended is long DNA having a length exceeding 500 kbp, a low loop is used from the viewpoint of preventing irreversible entanglement between the DNA molecule and the gel fiber. It is preferable that the frequency of the wave AC electric field is gradually increased.

In this case, the frequency of the low-frequency AC electric field is preferably gradually increased from 1 Hz to 20 Hz, preferably from 1 Hz to 10 Hz, from the viewpoint of preventing irreversible entanglement between the DNA molecules and the gel fibers. Is particularly preferred.

From the viewpoint of preventing the irreversible entanglement of the DNA molecules and the gel fibers, the rate of gradually increasing the frequency of the low-frequency AC electric field is preferably 0.01 to 1 HzZs, and more preferably 0.01 to 0.5 Hz / s. Preferably, 0.01 to 0.1 HzZs is particularly preferred.

When performing the nucleic acid molecule extension method of the present invention, from the viewpoint of preventing gel melting, all operations are performed at 0 to 80 ° C, preferably 10 to 35, more preferably 15 to 25 ° C. Is desirable.

The method for extending nucleic acid molecules of the present invention comprises disposing nucleic acid molecules in a gel, applying a low-frequency AC electric field to the nucleic acid molecules in the gel to extend the nucleic acid molecules, and then detecting the elongated nucleic acid molecules. The method further includes the step of collecting.

Means for observing the extension state of the nucleic acid molecule include an inverted fluorescence microscope, an upright fluorescence microscope, and a laser confocal microscope.

Means for detecting nucleic acid molecules include laser-induced fluorescence detector, ultraviolet / visible absorption detection, fluorescence detection, differential refractive index detection, thermo-optical detection, circular dichroism detection, electrochemical detection, and electrical conductivity. Detection and the like.

Means for recovering extended nucleic acid molecules include (1) re-melting the gel, (2) recovering the gel as it is, and (3) preparing a new gel around the gel containing the extended nucleic acid molecule. A method of recovering a new gel by an electrophoretic method, (4) a method of enzymatically or chemically cleaving the gel and then recovering the gel by an electrophoretic method, etc. These methods are performed by a conventional method.

The present invention further relates to a nucleic acid molecule extended by the extension method of the present invention. Such nucleic acid molecules are also effectively used for the development of single-molecule genome analysis technology that reads information from only one DNA molecule, and such single-molecule genome analysis technology. Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

Prior to the examples, the following devices were prepared and used in each example.

As shown in FIG. 1, a miniature cell for electrophoresis on an inverted fluorescence microscope was used for direct observation of the behavior of DNA molecules. Two Pt electrodes were placed on the miniature cell at a distance of 10111 or 0.5 cm. The AC electric field generated by the arbitrary waveform generator (1942, NF circuit design block) is increased to an appropriate intensity by the high-speed power amplification bipolar power supply (4020, NF circuit design block) and applied between the Pt electrodes. did. A 100x oil immersion objective lens (P 1 an — NEO FLUAR, NA = 1.3) and an inverted fluorescent microscope equipped with a high-pressure mercury lamp (Ax i 0 Vert 135 TV, Carrl Ze iss) were used. . The fluorescence image was observed using a SIT (silicon sensitized target) camera (C2400-08, Hamamatsu Photonics). The DNA image and electric field voltage were recorded directly to the memory of a personal computer using an image capture board (Himawari, Library 1). The maximum diameter and the center of gravity of the DNA molecule were calculated using image processing software (Cosmos 32, Library, Inc.). Example 1

15.5 pounds of cloves (165.6 kbp, manufactured by Futatsu Gene Co., Ltd.) were stained with a fluorescent dye, YOYO-1 (Molecular Probes Co., Ltd.). 1.0% by weight agarose gel (Agarose NA, manufactured by Amersham Pharmacia Biotech) (5 mm × 5 mm × 2 mm) was injected under a steady electric field (5 V / cm) for 5 minutes. To remove oxygen from the gel before observing the DNA, agarose gel was added to 4% (v / v) 2-mercaptoethanol, 2. It was subjected to a solution containing 3 mg / ml glucose, 0.1 mgZm1 glucose oxidase, and 0.01 SmgZm1 force codase.

FIG. 2A) shows a fluorescent image extending a T4 DNA molecule. Here, an AC electric field (10 Hz, 200 V / cm) was applied for a time of 0 to 70 seconds. FIG. 2B) shows the change in the maximum diameter of DNA [R, (^ m)]. Here, the maximum diameter refers to the longest diameter passing through the center of gravity of the target DNA fluorescence image. As shown in Fig. 2B), R, gradually increased and reached an asymptotic value after about 20 seconds. Here, the value of Ri fluctuates around 40 m, while the natural length of a double-stranded T4 DNA molecule is about 55 m. Due to the pore size heterogeneity of the agarose gel, the DNA molecules were sometimes trapped in such an intermediate state around 10 seconds in Figure 2B). During the extension process in the agarose gel, the DNA molecules repeatedly trapped and escaped in this intermediate state. After this trial, the DNA molecules could find an effective path through the agarose gel to reach the asymptotic maximum diameter. Example 2

The effect of electric field frequency on DNA molecule extension was analyzed. T4 DNA molecules were expanded under the same conditions as in Example 1 except that electric fields of various frequencies were applied. The results are shown in FIG. FIG. 3 shows the mean maximum diameter [R, (/ m)] as a function of electric field frequency. In Fig. 3, the black circles indicate the frequencies (1, 5, 6, 7, 8, 9, 1 0, 1 1, 1 2, 1 3, 1 5, 20, 30, 40, 50, 75, 100 (Hz) at 10 seconds with 1-5 different molecules at 10 seconds, then shows the averaged R between molecules, with open circles for 2 seconds (0.1 Hz) and 7 seconds (0.5 Hz) R, which was averaged between molecules after time averaging, is shown.

The motion of DNA molecules in a gel cannot be classified into frequencies such as those observed in polymer solutions (eg, linear motion, anti-resonance, stretching, orientation), but DNA molecules in motion JP frequency domain of FIG. 3 with an average maximum diameter of 1 ^ and the center of gravity of the X component G _x Can be established. Fluctuation level of G _x is, time-averaged R, small case than, DNA molecules, tube-like movement even during Agarosugeru (Ueda et al., (1 997) Stretching of Long DNA under Alternating Current Electric Fields in a Concentrated Polymer Solution, Polymer Journal, Vol.29. No.12. See ppl040-104 3). On the other hand, larger G _x variations indicate more complex movements than tube-like movements.

Figure 4 A), B) and C) shows 1 Hz, the time course of 10Hz and 1 00Hz (200 V / cm) R at each frequency, and G _x. Here, G _x has been recalculated so that the time average is zero. At 1 Hz, and G _x both fluctuate around the mean. G _x simply correlates with an external electric field, while R, varies in a more complex manner as shown in Figure 4A). At 10 Hz, both R, and are constant, as shown in Figure 4B). The DNA molecule extends under these conditions, but its position does not change. At 100 Hz, both R, and G _x are constant, as shown in FIG. 4C). Under these conditions, the DNA molecules are oriented but do not stretch. At low frequencies (around 0.1 Hz), linear motion of DNA molecules does not appear on agarose gels. Thus, it is understood that the average maximum diameter 1 ^ has a maximum value near 10 Hz. Example 3

The effect of electric field strength on the extension of DNA molecules was analyzed. Except that various electric field strengths were applied, the T4DNA molecule was elongated in the same manner as in Example 1. The results are shown in FIGS. FIG. 5A) shows the mean maximum diameter 1 ^ and the horizontal component G _x of the center of gravity of the T 4 DNA molecule at different electric field strengths (150 VZcm, 50 V / cm, and 250 V / cm). FIG. 5B) shows a fluorescence image corresponding to FIG. 5A). The bright spot at the left end of the DNA in Fig. 5B) indicates a coiled or bent structure. As shown in Fig. 6, the average maximum diameter of DNA molecules is almost proportional to the strength of the external electric field up to about SO OVZcm, and is 40 OV / cn! It gradually decreases in the region up to 60 OVZcm. The vertical dashed line in FIG. 6 indicates the boundary between the stretched and unstretched regions.

7) A), B) and C) show the time variation of the mean maximum diameter 1 ^ at 10 ^! 2 and the horizontal component G _x of the center of gravity of the T4DN A molecule. In the stretch region, and G _x both fluctuate slightly regardless of the frequency of the external electric field, as shown in Figs. 7A) and B). The average value for 10 seconds is 5 OVZcm each

= 36.4 ± 0.9 (/ m) and G _x = 0.0 ± 0.7 (zm). ! ! And ^ = 4 3.6 ± 1.1 (〃m) and G = 0.0 ± 2.4 (/ m). In essence, R, and G _x are constant in the stretch region. G _x variation

(0. and 2.4 / m) are 20 times smaller than the average maximum diameter (36.4 m and 43.6 ^ m) and do not disturb the stretched state of DNA molecules. On the other hand, in the case of 60 OVZcm, the variation in the magnitude of both and G is as shown in Figure 7C). The average value for 10 seconds is = 21.0 ± 4.4

(〃M) and G _x = 0.0 ± 9.9 (〃m), respectively. The standard deviation of G _x (9.9 fx m) is large enough to disrupt the conformation of the DNA molecule compared to the mean maximum diameter (2 1. O zm). Therefore, the present inventors can point out a qualitative difference between DNA behavior in a region of 45 OVZcm or less and DNA molecule behavior in a region of 450 V / cm or more. In high electric fields (50 OVZcm and 600 v / cm), DNA molecules travel a wide distance, comparable to the average largest diameter of DNA. As a result, the higher order structure of the DNA molecule is always disrupted at the agarose gel cross-link points. Therefore, in this region, the DNA cannot be stretched. Example 4

The effect of gel concentration on DNA molecule extension was analyzed. Agarose of various concentrations The T4DNA molecule was extended in the same manner as in Example 1 except that Sgel was used. Fig. 8 shows the results. FIG. 8 shows the dependence of the average maximum diameter of DNA on agarose concentration. The average maximum diameter of DNA is essentially constant over a wide range of 0.5-3% agarose concentrations. The pore size of the gel corresponding to this concentration is between 800 and 260 nm. At low concentrations (as low as 0.5%), the gels are very soft and cannot create a practical gel system for DNA extension. The dashed line in FIG. 8 indicates about 3.5 tzm, which is the average maximum diameter of T4 DNA in a 58% sucrose solution under the same electric field conditions. Thus, it should be noted that DNA molecules never extend in low molecular weight solutions. Therefore, it means that restriction of DNA molecule movement by the entanglement effect with gel fiber is necessary for DNA molecule extension. FIG. 8 shows that even the pore size of the 0.5% agarose gel (800 nm) is small enough to constrain the molecular motion of T 4 DNA to tube-like motion. Example 5

Stretching of Saccharomyces cerevisiae chromosomal DNA molecules was performed using low-frequency alternating electric fields of increasing strength and frequency. Saccharomyces cerevisiae chromosome DNA (225-2,200 kbp, BioRad Laboratories) was purchased as a gel plug. The gel is cut into small pieces (approximately 3 mm x 2 mm x 1 mm) with a razor and placed in 0.5 XT BE buffer containing 0.1 MYOYO-1 and 4% (vZv) 2-mercaptoethanol. Saved at 4 for 1 week. The gel slice was then incubated in a molten agarose solution on a mini-electrophoresis cell. After gelation of agarose, the agarose gel was treated with 4% (v / v) 2-mercaptoethanol, 2.3 mg / ml glucose, 0.1 mg / m1 glucose oxidase, and 0.018 mgZm1 The solution was added to a solution containing lipstick. Chromosomal DNA molecules were injected from a gel plug into agarose gel for extension under a 1 hour steady electric field (1-2 V / cm). Then replace the buffer in the reservoir with a new buffer Then, he began experimenting with DNA molecule extension.

As can be seen from Examples 1-4, the preferred conditions for extension of the T4 DNA molecule are 1% agarose gel, 10 Hz and 200 VZcm electric field strength. In addition to this condition, by gradually increasing the frequency and intensity of the electric field from the low frequency and low electric field intensity, the Saccharomyces cerevisiae chromosome DNA molecule is prevented while preventing irreversible trapping of DNA molecules on gel fibers. Stretched. First, a 1 Hz sine-wave AC electric field (I100IVZcm) is applied for 1 minute to orient the chromosomal DNA molecules in the direction of the electric field, and then the frequency is increased up to 10 Hz at a rate of 0.5 HzZs. The sine wave type AC electric field was applied for 1 minute while the electric field strength was gradually increased to 200 VZcm at the rate of I 10 I VZcm · s.

FIG. 9A) shows a fluorescence image of a Saccharomyces cerevisiae chromosome DNA molecule (about 285 kb P) when extended using a low-frequency AC electric field. This fluorescent image consists of three images, and the maximum diameter of the stretched DNA molecule is about 100 μm. Figure 9B) is a trace of the fluorescence image. The arrow in the “coil” part of FIG. 9B) indicates the coil part of the DNA molecule. This part is not fully extended. The “vent” arrow in Fig. 9B) indicates a bent structure, and such a structure may appear near the end of DNA. From FIGS. 9A) and B), it can be seen that a long-chain DNA molecule of about 285 kbp was successfully extended by the nucleic acid molecule extension method of the present invention. Industrial applicability

ADVANTAGE OF THE INVENTION According to the nucleic acid molecule extension method of this invention, it becomes possible to extend a nucleic acid molecule more easily, and it has the outstanding effect that the extended nucleic acid molecule can be collect | recovered and reused. .

Claims

The scope of the claims

1. A method for elongating a nucleic acid molecule, comprising the steps of placing a nucleic acid molecule in a gel and applying a low-frequency AC electric field to the nucleic acid molecule in the gel.

2. The method of claim 1, wherein the length of the nucleic acid molecule is 23-5700 kbp.

3. The method according to claim 1, wherein the frequency of the low-frequency AC electric field is 1 to 100 Hz.

4. The method according to any one of claims 1 to 3, wherein the intensity of the low-frequency AC electric field is I10I to I10000I (absolute value) VZcm.

5. The method according to any one of claims 1 to 3, wherein the intensity of the low-frequency AC electric field is gradually increased.

6. The method of claim 5, wherein the intensity of the low frequency alternating electric field is gradually increased from I 10 I VZcm to I 300 I (absolute value) V Zcm.

7. The method according to claim 5, wherein the rate at which the intensity of the low-frequency alternating electric field is gradually increased is I 1 l to l 100 I (absolute value) VZcm * s.

8. The method of claims 1 to 7, wherein the frequency of the low frequency alternating electric field is gradually increased.

9. The method of claim 8, wherein the frequency of the low frequency alternating electric field is gradually increased from 1 Hz to 20 Hz.

10. The method according to claim 8, wherein the rate at which the frequency of the low-frequency AC electric field is gradually increased is 0.01 to 1 HzZs.

11. The method according to any one of claims 1 to 10, further comprising a step of detecting and recovering the extended nucleic acid molecule.

1 2. A nucleic acid molecule elongated by the method according to any one of claims 1 to 11.