WO2009054564A1

WO2009054564A1 - Method for the synthesis of nucleic acid by using fragmented nucleic acids and n-primer

Info

Publication number: WO2009054564A1
Application number: PCT/KR2007/006070
Authority: WO
Inventors: Jae Sun Moon; Sung Uk Kim; In Gyu Hwang; Nam Soo Jwa; Suk Yoon Kwon
Original assignee: Korea Research Institute Of Bioscience And Biotechnology
Priority date: 2007-10-26
Filing date: 2007-11-28
Publication date: 2009-04-30
Also published as: KR100991724B1; KR20090042344A

Abstract

The present invention relates to a method for the synthesis of nucleic acid based on RNA fragmentation and N-primer which is prepared by using a fragmented RNA as a template, a method for constructing a cDNA library, a cDNA library constructed by said method, and a kit comprising N-primer for the synthesis of nucleic acid. According to the present invention, N-primer which may have the same complementarity for all regions of mRNA and various kinds of nucleic acids including mRNAs that are randomly fragmented by an artificial method are used for the synthesis of cDNAs or strands of complementary nucleic acids for every region. Thus, an overlap can be avoided and all kinds of cDNAs originating from mRNA or base sequences of the nucleic aids that are complementary to mRNA can be obtained.

Description

METHOD FOR THE SYNTHESIS OF NUCLEIC ACID BY USING FRAGMENTED NUCLEIC ACIDS AND N-PRIMER

Technical Field

[1] The present invention relates to a method for the synthesis of nucleic acid by using fragmented RNA and N-primer, a method for constructing a cDNA library, a cDNA library constructed by said method, and a kit comprising N-primer for the synthesis of nucleic acid. Background Art

[2] DNA synthesis has been used for various purposes in studies of a genetic engineering field. Except the synthesis of single stranded DNA such as oligonucleotide, most of DNA synthesis is carried out by an enzymatic method which uses a DNA polymerase.

[3] In order to synthesize cDNA from whole RNA or mRNA, a specific primer having a sequence complementary to a subject RNA or a primer having poly-T sequence have been used. cDNA can be synthesized when a base sequence is known for the subject RNA in a sample or a subject is an mRNA having a poly-A tail. However, except such cases, a subject RNA having unknown base sequence or a foreign RNA (e.g., RNA with a viral origin, etc.) cannot be converted to a cDNA.

[4] To overcome such limitation, Marcelo Bento Shares et. al. created a concept of "Normalization and Subtraction" [Marcelo Bento Shares et al., (1996) Genome Research. 6(9); 791-806] by which a probability for obtaining genetic information derived from RNAs having a relatively low copy number is enhanced by eliminating RNAs having a great number of copies in library. However, the above-described problem relating to the obtainment of genetic information that is actually expressed remains unsolved in this work.

[5] To overcome such problem, Andrew J. G. Simpson et. al. described the preparation of cDNA library in which full-length RNA and a randomly chosen primer were used to synthesize a first single-stranded cDNA and PCR was used to synthesize a second single-stranded cDNA in order to construct a library. By dosing so, more genetic information was obtained from the middle than the terminal regions of a gene that is actually expressed. However, in terms of a random obtainment of genetic information for the whole region of a gene with the same level, the primer used in said method is not a primer having completely random sequences. In addition, strong reliance on PCR still existed. Furthermore, because the second strand is synthesized by PCR according to said method, an insert contained in the resulting library is very short, thus it cannot be efficiently used for obtaining information regarding an expression sequence tag (hereinafter abbreviated as EST). Still furthermore, no suggestion has been made to appropriately control the length of inserts. Thus, at present time this method cannot be widely used.

[6] Meanwhile, thanks to the results obtained from Human Genome Project, information about human genome is now almost completely available. However, the number of human genes is assuned to be in the range of about 20,000-30,000 and still unclear. In addition, although almost more than 8 million ESTs have been established, genetic information on genes that are actually expressed is largely unidentified yet. In this connection, the method described in the present invention can be used for an efficient and almost complete obtainment of genetic information of every living organism and its economical value will be tremendous especially for humans. Disclosure of Invention Technical Problem

[7] Purpose of the present invention is to maximize efficiency for obtaining genetic information by introducing a new idea to a conventional method for constructing libraries, i.e., by avoiding an overlap and making a multiple determination possible even without using a mRNA sequence that is common between different living organisms or eukaryotes carrying different genomic information. Technical Solution

[8] In order to solve the problems described above, the present invention provides a method for the synthesis of nucleic acid based on RNA fragmentation and N-primer.

[9] Furthermore, the present invention provides a method for constructing a cDNA library based on RNA fragmentation and N-primer, and a cDNA library constructed by said method.

[10] Still furthermore, the present invention provides a kit comprising N-primer for the synthesis of nucleic acid.

Advantageous Effects

[11] According to the present invention wherein nucleic acids are synthesized by using N- primer and fragmented nucleic acids, an overlap can be avoided and the obtainment of base sequences corresponding to whole mRNA is possible, because instead of a terminal region of mRNA synthesis of cDNA starts at any region of mRNA with the same complementarity to them thanks to the characteristic of N-primer. As a result, a problem associated with a synthetic overlap which often occurs during the obtainment of ESTs can be overcome.

Brief Description of the Drawings

[12] Figure 1 is a photographic image of agarose gel electrophoresis for mRNA which was isolated from human liver and brain and sonicated.

[13] Figure 2 is a photographic image of agarose gel electrophoresis for first-strand cDNA and second-strand cDNA, which have been synthesized following the sonication of corresponding mRNAs isolated from human liver and brain.

[14] Figure 3 is a photographic image of 0.7% agarose gel electrophoresis wherein first- strand cDNA and second-strand cDNA, which have been synthesized and digested with Xhol restriction enzjme, were loaded to the gel to confirm the elution of the cDNA inserts having a size of 0.5- 1.5kb.

[15] Figure 4 shows position of the inserts carried by selected and sequenced clones relative to full-length cDNA sequence, wherein the data is plotted for two different methods for constructing a library.

[16] Figure 5 is a continuous representation of the data shown in Figure 4.

[17] Figure 6 shows the result for sequencing the nucleic acids that have been synthesized either by kribb method or by pnas method. It is confirmed from Figure 6 that, for pnas method, most of the inserts have a size less than 0.5kb while for kribb method, most of the inserts have a size the same or bigger than 0.5kb. Mode for the Invention

[18] In order to achieve the purpose of invention described above, the present invention provides a method for the synthesis of nucleic acids comprising following steps:

[19] fragmenting a RNA;

[20] synthesizing a first strand cDNA by using said fragmented RNA as a template and by annealing N-primer to said template; and

[21] adding a DNA poljmerase to said first strand cDNA to synthesize a second strand cDNA.

[22] The method for synthesizing cDNA suggested in the present invention is designed to obtain all the genetic information of an expressed gene by using an N-primer to prepare cDNA from a fragmented RNA, with the same chance for every region of the gene including two terminal regions and any region between them. Unlike conventional primers, an N-primer can have all kinds of possible combination of base sequences. Thus, it is expected that N-primers prepared according to the present invention are complementary to any sequence present in a sample. Due to such a characteristic, any RNA with any kind of base sequences present in a sample is expected to be converted to a corresponding cDNA. Therefore, nucleic acids can be synthesized from any kind of RNAs and DNAs of organisms including a fragmented mRNA without poly-A tail, rRNA, RNA and DNA from viruses or prokaryotes, etc. Further, the method of the present invention is remarkable in that it can be used to avoid an overlap of ESTs for obtaining genetic information of an expressed sequence of eu- karyotic cells. Still further, in addition to the avoidance of an overlap, the method of the present invention is also advantageous in that it can obtain an amplifying effect because mRNA is fragmented into many pieces and used for the synthesis of nucleic acid. Thus, the method of the present invention which uses an N-primer for the synthesis of nucleic acid is especially useful for following cases. [23] First, there has been a method to obtain entire genome sequence of a subject organism in order to have genomic information thereof. However, when it is difficult to identify a region having an actual function as a gene or there are other limitations such as a size of a genome, etc., sequences of genes that are expressed in a cell can be identified. However, the problem of such method is that due to single-pass sequencing a sequence of an identical region that is expressed from an identical gene (mainly 5 '-end or 3 '-end) is overlapped, making the method very inefficient for obtaining the entire nucleotide sequence of mRNA and posing a limitation for identifying many regions of the expressed gene. On the other hand, when RNA is cut into short fragments and nucleic acids are synthesized therefrom using an N-primer, cDNAs are synthesized randomly starting from all over the mRNA with the same complementarity to it instead of being synthesized from the end of mRNA, due to a characteristic of an N-primer. As a result, an overlap can be avoided and base sequences for entire mRNA can be obtained. When an mRNA is converted to a cDNA by using an N-primer, most of the synthesized cDNA appear to be the one synthesized from full-length mRNA using a primer with poly-T sequence. Thus, to correct such problem, a pure isolated mRNA is first cut physically or chemically and used for the synthesis of cDNA with an N-primer. Because the effect of an N-primer becomes more significant as the size of mRNA is bigger, it is believed that the method of the present invention can be essential for identifying a tissue-specific gene and obtaining an alternative splicing form which is required for full functioning of a gene, or for identifying a gene related to a disease and obtaining an alternative splicing form which is specific to a certain type of a disease. [24] Second, for a detection of pathogens, advantages of using an N-primer can be considered in various ways. In order to detect many kinds of pathogens in interest using a chip, all of the nucleic acids present in a sample is labeled with a fluorescent compound, etc. and then treated onto the chip. In this case however, if a pathogen present in a sample has not been identified so that a base sequence of its nucleic acids specific for such pathogen is not known, chip-based detection is impossible per se. But by using an N-primer instead, all the nucleic acids can be labeled with a fluorescent compound, thus a chip can be successfully used even when there are lots of genes to be detected. Further, without cutting entire RNA or mRNA by a physical or chemical method, various kinds of pathogens can be detected simultaneously thanks to the randomness of an N-primer. Especially for an oligonucleotide DNA chip, when nucleic acids extracted from a subject are poor in their quality or quantity, RNA can be first cut physically or chemically and then used. By doing so, an amplifying effect can be achieved, thus enabling the obtainment of a better result. In addition, for dT primer or a reverse primer synthesis of nucleic acids occurs only from 3 '-direction of mRNA. For N-primer however, it can occur from both directions. Thus, even from a negative strand present in a sample nucleic acids can be synthesized, which also contributes to the effect of amplifying a subject gene as described above.

[25] Third, when an instrument such as PCR, etc. is used for identifying a certain genetic material comprised in a sample, the entire sample is usually wasted when such material is not successfully identified. However, when nucleic acids are synthesized by using an N-primer, the sample can be kept for a while and be used again for a later experiment when something interesting is found out for that genetic material. For example, there can be a situation that a condition similar to viral disease occurs to a hot pepper and all of the diagnostic determination is made for the hot pepper in relation to the every virus that has been known to infect hot pepper but no virus is detected therefrom. In such case, a hot pepper sample of which nucleic acids are prepared using a reverse primer or an oligo dT primer will be wasted. On the other hand, a hot pepper sample of which nucleic acids are prepared using an N-primer can be preserved and used again later, i.e., when a new virus is found for attacking a hot pepper, the preserved sample is brought back and used for a test which determines the infection of a hot pepper with the new virus. Such characteristic of an N-primer can be advantageously used.

[26] The method of synthesizing nucleic acid according to the present invention includes a step of fragmenting RNAs. RNAs can be anyone of mRNA, rRNA without poly- A tail, or RNA from viruses of prokaryotes but are not limited thereto. When an mRNA is converted to a cDNA actually using an N-primer, most of the synthesized cDNA appear to be the one synthesized from full-length mRNA using a primer with poly-T sequence. Thus, to correct such problem, a pure isolated mRNA is first cut physically or chemically and used for the synthesis of cDNA with an N-primer. The fragmentation of RNAs can be carried out by sonication but not limited thereto. Any physical or chemical method which can fragment RNAs can be used. It is preferred that RNA fragments obtained by sonication has a size within the range of 0.5- 1.5kb. When the size of RNA fragments is not within said range, an effective sequencing result cannot be obtained compared to the size that can be interpreted by single -pass sequencing of a current technology. Thus, to obtain the same amount of information, not only an excessive number of clones have to be sequenced but also many problems may arise during the assembling of sequencing results. In addition, if a fragment is bigger than 1.5kb, it cannot be interpreted by a single-time sequencing from both termini.

[27] RNAs that are used as a template for the method of the present invention can be isolated or produced from any kind of sample carrying nucleic acids. Non-limiting examples of a sample which can carry nucleic acids includes the following; samples from an organism including whole blood, blood serum, white cell layer (buffy coat), urine, faces, cerebrospinal fluid, semen, saliva, tissue (e.g., cancerous tissue or ljmph node) and cell culture (e.g., mammalian cell culture or bacterial cell culture), samples comprising nucleic acids including, viroid, virus, bacteria, fungus, yeast, plant and animal, samples that are suspected to be contaminated or infected with microorganisms such as bacteria or virus (e.g., food or biological preparation), and samples that may comprise an organism such as soil and water, etc. Nucleic acids can be further prepared from said materials by shaking or stirring them with a glass bead or French press, cell lysis using a detergent and sonication, etc.

[28] The method for the synthesis of nucleic acid according to the present invention includes a step of synthesizing a first strand cDNA by annealing an N-primer to the above-described fragmented RNAs as a template. Any conventional method used in the pertinent art can be employed for synthesizing a first strand cDNA. For example, the first strand cDNA can be synthesized by using a reverse transcriptase and an inhibitor for RNase block ribonuclease, etc. Examples of reverse transcriptase includes those originating from various sources, for example, avian myeloblastosis virus- derived virus reverse transcriptase (AMV RTase), murine leukemia virus-derived virus reverse transcriptase (MMLV RTase) and Rous-associated virus 2 reverse transcriptase (RA V-2 RTase).

[29] On the other hand, only a specific kind of N-primer can be used as a primer in the present invention. N-primer is a primer which can have any of G, A, T and C bases for its respective position for nucleotides. For example, for an N-primer consisting of thirty nucleotides, the nurber of possible conϋnation is 4 ³⁰ for such primer. Preferably an N-primer consists of 5 to 50 nucleotides. More preferably, it consists of 25 to 30 nucleotides. When the length of an N-primer is not within said range, it is possible that the efficiency for synthesizing a desired nucleic acid can be lowered.

[30] The method for the synthesis of nucleic acid according to the present invention further comprises a step of synthesizing a second strand cDNA by adding a DNA polymerase to a first strand cDNA as synthesized above. Any conventional method used in the pertinent art can be employed for synthesizing a second strand cDNA. For example, the second strand cDNA can be synthesized by using RnaseH or DNA polymerase I, etc. DNA polymerase used herein means an enzyme which can synthesize a new DNA strand by using one existing DNA strand as a template. Non- limiting examples of DNA polymerase include Pol I-type DNA polymerase (e.g.: Escherichia coli DNA polymerase I, Klenow fragment and Taq DNA polymerase), α- type DNA polymerase (e.g.: DNA polymerase from Pyrococcus furiosus (Stratagene), VENT DNA polymerase (New England Biolabs), KOD DNA polymerase (Toyabo) and DEEP VENT DNA polymerase (New England Biolabs)) and non-α-, non-Pol I- type DNA polymerase (e.g.: DNA polymerase described in WO 97/24444). As a DNA polymerase having a strand displacement activity, examples include DNA polymerase from thermophilic bacteria of Bascillus species such as Bacillus caldotenax and Bacillus stearothermophilus, and their variant of which 5'→3' exonuclease activity has been deleted. DNA polymerase having a strand displacement activity further includes a DNA polymerase which has a strand displacement activity but not 5'→3' exonuclease activity, for example Klenow fragment. Without any limitation, DNA polymerase can be a mixture of various kinds of DNA polymerases such as a mixture of a DNA polymerase with a strand displacement activity and a DNA polymerase without it.

[31] The term "strand displacement activity" used herein indicates an activity for displacing a strand, i.e., displacing a DNA strand and copying DNA based on a nucleotide sequence as a template while the complementary strand that is annealed to the template strand is separated. In addition, a DNA strand which is separated from the nucleotide sequence as a template that has been obtained by strand displacement is referred to as "displaced strand" in the present specification. [32] In order to achieve another purpose of the invention, the present invention provides a method for constructing a cDNA library comprising the following steps:

[33] fragmenting a RNA;

[34] synthesizing a first strand cDNA by using said fragmented RNA as a template and by annealing N-primer to said template; and

[35] adding a DNA polymerase to said first strand cDNA to synthesize a second strand cDNA.

[36] The fragmentation of RNAs can be carried out by sonication. It is preferred that

RNA fragments obtained by sonication has a size within the range of 0.5- 1.5kb. Preferably N-primer consists of 5 to 50 nucleotides. More preferably, it consists of 25 to 30 nucleotides. When the length of an N-primer is not within said range, it is possible that the efficiency for synthesizing a desired nucleic acid can be lowered. Detailed information regarding the fragmentation of RNAs, N-primer and DNA polymerase, etc. are the same as those given above.

[37] For the method to construct a cDNA library according to the present invention, other general steps as follows can be additionally included but are not limited thereto; i.e., cDNA termini blunting, treatment with a restriction enzyme such as EcoRI, ligation with an adaptor for a restriction enzyme and phosphorylation of termini, treatment with a restriction enzyme such as Xhol enzyme, cDNA separation by gel elution, ligation between cDNA insert and a vector, packaging reaction and mass excision, etc. Said general steps can be carried out by using a method which is publicly known to a person in the pertinent art.

[38] The present invention further provides a cDNA library that is constructed based on said method for constructing a cDNA library.

[39] In order to achieve yet another purpose of the present invention, a kit for synthesizing nucleic acids comprising N-primer and other reagents necessary for the nucleic acid synthesis is provided. Preferably, an N-primer consists of 5 to 50 nucleotides. More preferably, it consists of 25 to 30 nucleotides. When the length of an N- primer is not within said range, it is possible that the efficiency for synthesizing a desired nucleic acid can be lowered. Other reagents necessary for the nucleic acid synthesis may include reverse transcriptase, DNA polymerase, and Rnase H, etc. but are not limited thereto. Said kit of the present invention may comprise a guidebook. "Guidebook" is a printed material which explains a method for how to use a kit, for example, a method for preparing a solution of reagents required for the nucleic acid synthesis, and a suggested reaction condition, etc. The guidebook also can include a handbook in a form of pamphlet or brochure, a label attached to the kit, and an explanation described on a package comprising the kit. Further, the guidebook includes any information known from or given in electronic media such as internet.

[40] The present invention will now be described in greater detail with reference to the following examples. However, it is only to specifically exemplify the present invention and in no case the scope of the present invention is limited by these examples.

[41] Examples

[42] Experiments that have not been specifically described herein below can be carried out by any molecular biological methods that are well known to a skilled person in the pertinent art.

[43] Example 1: Construction of cDNA library using N-primer

[44] For N-primer cDNA library, Stratagene ZAP-cDNA Qgapack m Gold Cloning Kit

(cat. # 200450) was used and experiments were carried out according to the manufacturer's protocol. By using human liver and brain mRNA supplied by Clontech, cDNA library was constructed.

[45] 1. Sonication of mRNA

[46] 5^g of mRNA was adjusted to total volune of 400 //# and subjected to sonication for

6 sec using a sonicator manufactured by Sanies & Materials Inc. To the sonicated mRNA, 100% ethanol (8OO/i6) and 3M sodiun acetate (4O/i6) were added and mixed well by inverting the tube. Precipitation was then carried out for 1 hr at -2O⁰C. The resulting precipitates were centrifuged at 4⁰C, 13000rpm for 30 min. Except the mRNA precipitated at the bottom, supernatant was removed. To the precipitate, 70% ethanol (1.2ml) was added to wash out impurities. Following the centrifuge at 4⁰C, 13000rpm for 10 min, supernatant was removed and the resulting well-dried mRNA was solubilized by adding sterilized water which has been treated with DEPC. Figure 1 is a photographic image of agarose gel electrophoresis for mRNA which was isolated from human liver and brain. From 20 //# of the final mRNA that has been prepared by the above-described sonication, OAβtt (100ng total) of mRNA was taken and loaded to 1% agarose gel. As it is shown in Figure 1, it was confirmed that most of RNAs were in the range of 0.5- 1.5kb.

[47] 2. Synthesis of first-strand cDNA

[48] IQx first-strand buffer (5μ£), first-strand methylnucleotide mixture (3μ£), Xhol-N25 primer (5'-GAGAGAGAGAGAGAGAGAGAAGCTTCTCGACN _(Z)-3' primer; SEQ ID No: 1, 9μg), and RNase block ribonuclease inhibitor (4OU//i6; lβ&) were all added to the sonicated mRNA as prepared above. Following gentle mixing, the reaction was carried out at 37³C for 10 min. Then, Strata script RTase (50U/ βΑ; 2.7SiA) was added to the sample and the reaction was carried out again at 37³C for 10 min. Subsequently, cDNA was synthesized at 42⁰C for 50 min.

[49] 3. Synthesis of second-strand cDNA

[50] First-strand cDNA synthesized above and reactants to be used for the synthesis of second-strand cDNA were kept on ice for 5 min to lower their temperature below 16⁰C. Then, 10x second-strand buffer (20/iβ), second-strand dNTP mixture (S ≠), sterilized water (107.5μ£) and first-strand cDNA sample (45μ£) were all mixed together and RNase H (1.5U//i6) and DNA pol}meraseI(9U//£6) were further added thereto. Following gentle mixing, the reaction was carried out 16⁰C for 2 hrs and 30 min. When the sample was taken out at the end of the reaction, caution has to be taken to put it immediately in ice so as to prevent a formation of hairpin structure. Figure 2 is a photographic image of agarose gel electrophoresis for first-strand cDNA and second- strand cDNA, which have been synthesized following the sonication of corresponding mRNAs isolated from human liver and brain, as described above. Each of 10% of the synthesized first-strand cDNA and second-strand cDNA had been loaded to 1% agarose gel. As it was shown in Figure 1, it was confirmed that cDNA having an appropriate size was synthesized.

[51] 4. cDNA termini blunting

[52] To the synthesized cDNA sample above (200/^β), blunting dNTP mix (23 /i6) and cloned pfu DNA poljmerase (2.5U//i2; 2βX) were added and then immediately mixed using vortex. The reaction was carried out at 72⁰C, for 30 min exactly. Then, a mixture of phenol : chloroform (1:1, pH 7-8; 2OO/i6) was added and mixed well using vortex. The mixture was centrifuged for 2 min with the maximum speed. Supernatant was transferred to a new tube and chloroform with the same volume as the supernatant was added, mixed by vortex, and then centrifuged for 2 min with the maximun speed. Resulting supernatant was again transferred to a new tube and 100% ethanol (400 //#) and 3M sodium acetate (2O/i6) were added and mixed well. After keeping the sample at -2O⁰C for overnight, it was centrifuged at 4⁰C, 15000rpm for 60 min. Supernatant was removed and 80% ethanol (5OO/i6) was added. Then, cDNA precipitated at the bottom of the tube was observed. Without any mixing, centrifuge was carried out at room temperature for 2 min. Ethanol was removed and the sample was left to dry for 10 min. Then, EcoRI adapter (9β&) was added and the sample was solubilized at 4⁰C for 2 hrs.

[53] 5. Ligation of ECoRIadapter and terminal phosphorylation [54] To a prepared sample, IQx ligase buffer (1/ig), 1OmM rATP (1/ig) and T4 DNA ligase (4U/βΑ; iβΑ) were added and a ligation was carried out at 4⁰C for 2 days. Two days later, the enzjmes were inactivated by heating at 7O⁰C for 30 min and the sample was left at room temperature for 5 min. Then, 10x ligase buffer (lβ&), 1OmM rATP (2β&), T4 polynucleotide kinase (5U//i6; 1μ£) and sterilized water (5β&) were added to ligate the adapter at 37⁰C for 30 min. Following the inactivation process at 7O⁰C for 30 min, the sample was left at room temperature for 5 min and used for next step.

[55] 6. Xholenzjme treatment

[56] In order to digest the phosphorylated cDNA strand with Xholrestriction enzjme,

Xhol buffer supplement (Ε≠), XhoI(40U//i6; 3≠) and the phosphorylated cDNA sample (22/i2) were added and the reaction was carried out 37⁰C for 1 hr and 30 min. Then, IQx STE buffer (5βΑ) and 100% ethanol (125μ£) were added and the mixture was kept at -2O⁰C for overnight. Next day, the sample was centrifuged at 4⁰C, 14000rpm for 1 hr. Supernatant was removed and the remaining residue was dried. Ix STE buffer (14/i6) was added to solubilize the dried cDNA.

[57] 7. Isolation and extraction of cDNA using gel permeation

[58] Sample was loaded to 0.7% agarose gel and the electrophoresis was carried out for

70 min under 25V. Then, only the cDNAs having a size of 500bp~ 1.5kb were excised and subjected to an extraction using QIAquick gel extraction kit (Qiagen, cat.28706). To the extracted cDNA, 100% ethanol (8O/i6) and 3M sodium acetate (4μA) were added and mixed well. After keeping the sample at -2O⁰C for one hour, it was centrifuged at 4⁰C, 15000 rpm, for 60 min. Supernatant was removed and 70% ethanol (200 /i6) was added. The sample was again centrifuged at room temperature for 2 min, and the remaining ethanol was all removed. After drying, the sample was soulbilized in sterilized water (4.5μ£). Figure 3 is a photographic image of 0.7% agarose gel electrophoresis wherein first-strand cDNA and second-strand cDNA, which have been digested with Xhol restriction enzjme, were loaded to elute their cDNA inserts. As it is shown in Figure 3, it was confirmed that cDNA having an appropriate size was synthesized.

[59] 8. Ligation of cDNA insert and vector

[60] To a prepared sample (3.5fd), Uni-ZAP XR vector (Iμglβt, Ifd), IQx ligase buffer

(0.5/ig), 1OmM rATP (pH7.5; 0.5/ig) and T4 DNA ligase (4U//ig; 0.5/ig) were added and mixed. Ligation was carried out at 4⁰C for 2 days.

[61] 9. Packaging reaction

[62] When a packaging extract, which had been stored at -8O⁰C, started to melt on ice, 4βΑ of the prepared sample was added thereto immediately. During the addition, the mixture was gently swirled by a using a tip to prevent incorporation of air. Centrifuge was carried out quickly for 2 to 3 sec and the sample was left at 22⁰C for 2 hrs. Following the addition of SM buffer (5OO/i6) and chloroform (2O/i6), the mixture was gently shaken and then simple centrifuge was carried out to precipitate residuals. Only the supernatant was taken to a new tube and kept at 4⁰C. Thus-prepared packaged sample and XLl-Blue MRF' cell, of which OD₆₀₀had been adjusted to 0.5 by using 1OmM MgSO₄, were admixed to each other and the reaction was carried out at 37³C for 15 min. Consequently, the resultant was added to NZY top agar mediun comprising X-gal and IPTG and then poured over the NZY agar medium. After culturing at 37³C for 12 hrs, cell titer was determined.

[63] 10. Mass excision

[64] The packaged sample obtained above, XLl-Blue MRF' cell and ExAssist helper phage were admixed to each other and the reaction was carried out at 37³C for 15 min. Then, 20 ml of LB broth with supplements was added thereto and the cells were cultured at 37⁰C for 3 hrs, while shaking them at 200rpm. Following the heat treatment of the cells at 65⁰C for 20 min for the lysis of the cells, the culture solution was centrifuged with lOOOg for 10 min to remove the precipitates. Only the supernatant was transferred to a new tube and admixed with SOLR cells for the reaction at 37³C for 15 min. To LB-ampicillin plate comprising X-gal and IPTG, an adequate amount of the cells were plated and then cultured at 37³C for overnight. Finally, only the white colonies were selected for further culture and DNA was extracted therefrom by using RBC hiyield miniprep kit.

[65] Example 2: Comparison between the method for synthesizing nucleic acid of the present invention and prior art method

[66] From the library constructed from the nucleic acids which have been synthesized according to the method of the present invention (hereinafter, referred to as 'kribb method'), clones were freely selected and their nucleotide sequence was compared to that of nucleic acid synthesized by prior art method [Andrew J. G. Smpson etal., (2000) Proc. Natl. Acad. Sci. USA 97(7): 3491-3496; hereinafter, referred to as 'pnas method'), in terms of their size and position relative to full-length RNA. With respect to 5 and 3 sequences, sequencing results for ESTs that had been freely extracted from the information made public by National Center for Biotechnology Information (NCBI) were extracted and used for the comparative analysis. According to pnas method, a single strand cDNA is synthesized by using full-length RNA and a randomly chosen primer and second-strand cDNA is synthesized by using PCR, thus providing a library. Detailed process of pnas method is the same as those described by Andrew J. G. Smpson, et. al.

[67] Figure 4 and Figure 5 show position of the sequences of the clones relative to full- length cDNA sequence, wherein said clones had been randomly selected from the library and the sequencing results were compared to the sequence of the full-length cDNA. As it is shown in Figure 4, the results obtained from the sequencing of the information made public by NCBI in 5 and 3 direction and those obtained from pnas method were also randomly extracted for the comparison with the results of the present invention. According to the sequencing results for 5 region, it was found that most of the sequences are present at front 20% of the full-length cDNA. On the other hand, according to the sequencing results for 3 region, it was found that most of the sequences are present at terminal 20% of the full-length cDNA. However, with respect to kribb method and pnas method, they are present almost uniformly over the full- length cDNA sequence. As such, it is found that nucleic acids synthesized either by kribb method or pnas method cover almost the entire sequence of the full-length cDNA. For such reason, the more information on EST is produced by a widely-used conventional method for constructing a library, the more repetitive information is generated inefficiently. In order to overcome such problems, studies have been made to obtain full-length cDNA and then sequence it. However, information regarding an alternative splicing form that is related to tissue specificity or certain diseases cannot be obtained through such approaches. On the other hand, according to kribb method of the present invention, inefficient generation of repetitive information can be avoided and information regarding an alternative splicing form that is related to tissue specificity or certain diseases can be easily obtained.

[68] Figure 6 shows size distribution of the inserts compared between kribb method and pnas method, wherein the nucleotide sequence of the inserts of clones, which had been freely selected from the libraries constructed according to either kribb method or pnas method, was determined by single-pass sequencing to obtain the size of the inserts. X- axis of Figure 6 corresponds to the length of the nucleic acid that has been sequenced. Y-axis of Figure 6 corresponds to the number of clones which have the corresponding size of nucleic acids. As it is shown in Figure 6, most of the sequenced nucleic acids which had been synthesized by pnas method are the same or less than 400bp long, while most of the sequenced nucleic acids which had been synthesized by kribb method are in the range of 600-800bp. Presumably, most of the nucleic acids syn- thesized by pnas method are inefficiently short due to a limitation of PCR method and because cDNA is synthesized by using random primers it is highly likely that mRNA having a certain sequence is not transformed into a corresponding cDNA. Further, since the inserts are extremely short, many problems may arise for an assembly of ESTs and a huge cost will be required for their sequencing, therefore presenting an inefficiency problem that cannot be easily overcome. On the other hand, when a library is constructed by using nucleic acids that are synthesized according to kribb method of the present invention, size of the inserts can be controlled to their most efficient level depending on each circumstance. In addition, unlike pnas method, even when sequences for all the genes to be expressed are different, every mRNA can be successfully transformed to a corresponding cDNA by N-primer. As a result, the method of the present invention is advantageous in that it can solve all the problems that are associated with conventional methods for constructing a DNA library.

Claims

[I] A method for the synthesis of nucleic acids comprising following steps: fragmenting a RNA; synthesizing a first strand cDNA by using said fragmented RNA as a template and by annealing N-primer to said template; and adding a DNA poljmerase to said first strand cDNA to synthesize a second strand cDNA.

[2] The method for the synthesis of nucleic acids according to Claim 1, characterized in that said RNA fragmentation is carried out by sonication.

[3] The method for the synthesis of nucleic acids according to Claim 2, characterized in that said fragmented RNA has a size of 0.5- 1.5kb.

[4] The method for the synthesis of nucleic acids according to Claim 1, characterized in that said N-primer has 5-50 nucleotides.

[5] The method for the synthesis of nucleic acids according to Claim 1, characterized in that said RNA is mRNA, rRNA having no polyA tail, or viral or prokaryotic RNA.

[6] A method for constructing a cDNA library comprising following steps: fragmenting a RNA; synthesizing a first strand cDNA by using said fragmented RNA as a template and by annealing N-primer to said template; and adding a DNA poljmerase to said first strand cDNA to synthesize a second strand cDNA.

[7] The method for constructing a cDNA library according to Claim 6, characterized in that said RNA fragmentation is carried out by sonication.

[8] The method for constructing a cDNA library according to Claim 7, characterized in that said fragmented RNA has a size of 0.5- 1.5kb.

[9] The method for constructing a cDNA library according to Claim 6, characterized in that said N-primer has 5-50 nucleotides.

[10] A cDNA library constructed by a method according to anyone of Claim 6 to

Claim 9.

[I I] A kit for the synthesis of nucleic acids comprising N-primer and other reagents required for the synthesis of nucleic acids.

[12] The kit according to Claim 11, characterized in that said N-primer has 5-50 nucleotides.