This application claims priority to and the benefit of Korean Patent Application No. 10-2008-0035772, filed April 17, 2008, the disclosure of which is incorporated herein by reference in its entirety.

The present invention relates to a method for constructing a viral genomic library from mixed states in which the viruses are mixed with the infected hosts, and relates to a method for carrying out genome project by screening only the viral genomic library.

Recently, most genomic projects are carried out by shotgun method, but according to purposes, mapped clone methods by constructing genomic library are also used. There are BAC (Bacterial Artificial Chromosome), Cosmid, Fosmid in the constructing genomic library.

Selection of target substances from the environment is the most important thing whether it is shotgun method or mapped clone method. But for the infectious virus, it is not easy to extract or cultivate, so it takes a lot of time to study of genome.

Insect biotechnology can be vastly applied in a plurality of fields, such as health care, agriculture and environments protection. In recent years, more countries have paid more attention to developing and industrializing insect genome resources. Further, some of most important biotechnological advances over the past few years have been achieved in association with genetics.

In genetics, using genomic clones has been of crucial importance in various ways.

A fosmid library is usually used as a backbone of a sequencing project for analysis of genomic clones. A fosmid library can be constructed by using the following processes of: acquiring environmental samples; extracting genomic DNA fragments from the environmental samples; purifying the DNA fragments; trimming the purified DNA fragments; performing size selection, concentration and ligation to copy control fosmid vectors; and transforming, arraying and replicating the DNA fragments.

Among them, the extraction of genomic DNA fragments is one of the most essential parts in constructing a fosmid library.

Pure extraction and cultivation are prerequisite for studying the genome of infected virus. But these processes are very complex and not easy, so it takes a lot of time to begin to study of genome.

However, the conventional method for extracting genomic DNA fragments from environmental samples has been performed in a complex way. For example, cells of the environmental samples need to be kept in a saline solution from being lysed. Then, the cells are separated from the suspended solution by spinning them in a centrifuge. After cell pellets are acquired, a lysis buffer is added to the cell pellets in order to split the cells open (the DNA must be released from the nucleus). The lysis buffer contains soap (to break apart the fatty membranes), salts and ions (to increase the osmotic pressure outside the cells and help break apart the membranes), and buffers (to maintain pH of the solution). Finally, the cells are incubated in a hot water bath in order to denature cytoplasmic enzymes which break apart the DNA.

Furthermore, the conventional method has a drawback in that a number of environmental samples are required in order to acquire effective and well-directed DNA samples available for the DNA sequence analysis.

1. Generating fosmid library with the states in which the viruses and the infected hosts are mixed. (Same with the usual method)

But after PFGE of the genomic DNA is separated from the infected hosts, generating library is performed with the 100 ~ 150 kb sample corresponding to genome size of virus.

2. Nucleotides terminal sequencing of the generated library (for 96 clones bidirectionally)

3. Using bioinformatics

a. construction of virus amino acid sequence data base (extract only the virus amino acid among sequence publicated in NCBI)

b. construction of viral genome database similar with the target virus.

c. construction of insect (or host) specific sequence data base (extract only the insect (host) sequence publicated in NCBI)

d. comparison the sequence of the clone with two database (virus and insect) using LOCAL BLAST (basic local alignment search tools) and characterization whether the insert of the clone is of the insect or of the virus.

e. making minimum tiling path by comparing the clones in which virus genome inserted with the similar virus genome sequence (tBLASTx)

f. constructing shotgun library by hydroshearing of selected clones and sequencing.

g. assemblying and finishing genome sequence by PhredPhrap software and primer walking.

It is, therefore, one object of the present invention to provide a method for constructing a fosmid library of Pieris rapae for enabling genomic DNA sequences of a Pieris rapae virus to be analyzed with fewer Pieris rapae samples without having to separate a Pieris rapae virus from Pieris rapae .

However, the objects of the present invention are not limited to the foregoing.

In accordance with the present invention, genomic DNA sequences of a Pieris rapae virus are analyzed without separating a Pieris rapae virus from Pieris rapae , so that a time required for DNA sequence analysis can be significantly reduced and the experiment can be performed only with much fewer Pieris rapae samples.

The above and other objects and features of the present invention will become apparent from the following description of preferred embodiments given in conjunction with the accompanying drawings, in which:

Fig. 1 is a flow chart showing a method for constructing a fosmid library of Pieris rapae in accordance with the present invention;

Fig. 2 shows an experimental result in accordance with the present invention, exhibiting HMW DNA embedded agarose plugs of Pieris rapae are checked by using PFGE, wherein the plugs are partially digested by an enzyme mixture following pre-electrophoresis;

Fig. 3 shows an experimental result of PCR by using PFGE in accordance with the present invention, wherein PCR is performed on 5 primers designed from the nucleotide sequence of a Pieris rapae virus and the size of the PCR product is the same as the expected size of the nucleotide sequence;

Fig. 4 shows an experimental result in accordance with the present invention, indicating the DNA of the Pieris rapae virus corresponding to 125 kb is separated from Pieris rapae DNA embedded agarose molds;

Fig. 5 shows an experimental result in accordance with the present invention, indicating the concentration of DNA that has been collected by PFGE is determined by using a spectrophotometer;

Figs. 6 and 7 are experimental results in accordance with the present invention, showing data prior to and posterior to mapping fosmid clones based on the result of blast nt & nr performed on granulovirus and 9 kinds of Pieris rapae, respectively;

Fig. 8 shows an experimental result in accordance with the present invention, representing the size of the 4 selected fosmid clone DNAs measured by NotI restriction enzyme digestion of fosmid clone DNAs; and

Fig. 9 is a schematic diagram including a flow chart of shot-gun library construction.

In accordance with one aspect of the present invention, there is provided a method for constructing a fosmid library of Pieris rapae, which comprises the steps of: separating genomic DNA from a Pieris rapae virus and Pieris rapae; washing the genomic DNA; preparing genomic DNA embedded agarose plugs; performing electrophoresis and dialysis on the agarose plugs; partially digesting the agarose plugs; separating DNA of the Pieris rapae virus from DNA of the Pieris rapae; mapping fosmid clones of the separated DNA of the Pieris rapae virus; and shotgun library construction of the mapped fosmid clones.

In the following detailed description, reference is made to the accompanying drawings that show, by way of illustration, specific embodiments in which the invention may be practiced. These embodiments are described in sufficient detail to enable those skilled in the art to practice the invention. It is to be understood that the various embodiments of the invention, although different, are not necessarily mutually exclusive. The following detailed description is, therefore, not to be taken in a limiting sense, and the scope of the present invention is defined only by the appended claims that should be appropriately interpreted along with the full range of equivalents to which the claims are entitled.

Then, experiments performed for better understanding the present invention will be described in detail as follows, which are set forth to illustrate, but are not to be construed to limit the present invention.

First, a fosmid library of Pieris rapae is constructed in order to analyze genomic DNA sequences of a Pieris rapae virus without separating a Pieris rapae virus from Pieris rapae.

Pieris rapae used for the present invention is scientifically classified into a species of P. rapae, a genus of Pieris, a tribe of Pierini, a family of Pieridae, a superfamily of Papilionoidea, an order of Lepidoptera, a class of Insecta, a phylum of Arthropoda, and a kingdom of Animalia, which was raised in a cage for insects for a short time in order to perform experiments of the present invention.

Next, specific methods for constructing a fosmid library of Pieris rapae in accordance with present invention will be described in further detail, with reference to the accompanying drawings.

Fig. 1 is a flow chart showing a method for constructing a fosmid library of Pieris rapae in accordance with the present invention.

Referring to Fig. 1, the fosmid library in accordance with the present invention may be constructed by the following steps of: separating genomic DNA from a Pieris rapae virus and Pieris rapae; washing the genomic DNA; preparing genomic DNA embedded agarose plugs performing electrophoresis and dialysis on the agarose plugs; partially digesting the agarose plugs; separating DNA of the Pieris rapae virus from DNA of the Pieris rapae; mapping fosmid clones of the separated DNA of the Pieris rapae virus; and shotgun library construction of the mapped fosmid clones.

Hereinafter, it will be described in more detail how each step is performed.

(1) Separation of nuclei from Pieris rapae

Samples of Pieris rapae that have been caught and raised in a cage for insects for a short time are completely ground by a tissuelizer in order to separate the nuclei from the whole body from which guts have been removed. Then, 1.250 g of the ground samples is put in a tube onto which 20 ml of a suspension solution is added. The remainder of the ground samples is kept at -80 Celsius degrees. The completely suspended samples in the suspension solution are centrifuged at 5,000 rpm for 10 minutes at 4 Celsius degrees to remove cell debris from the solution. Then, the solution is re-added onto the tube so as to be adjusted to a total volume of 1 ml.

(2) Preparation of HMW DNA plugs embedded in agarose

High molecular weight (HMW) DNA is considerably vulnerable to a mechanical shearing, so that HMW DNA may inevitably get damaged in the nuclei lysis process. To prohibit HMW DNA from being damaged in the nuclei lysis process, the separated nuclei are put into an agarose gel. 1 % InCert agarose (BMA) is molten and then kept at 45 Celsius degrees in a water bath. The nuclei are warmed for 5 minutes at 45 Celsius degrees in a heating block set and mixed well with the molten agarose. Then, the mixture is poured into a plug mold (BioRad) on the ice and allowed to solidify for 1-2 hours. Next, the agarose plugs are put into 50 ml of proteinase K lysis buffer (0.5 M EDTA, 1 % N-laurosylascosine, 1 mg of protease K/ml) and are incubated for up to 24 hours at 50 Celsius degrees. After the proteinase K lysis buffer is removed from the agarose plugs, the lysis process is additionally performed on the agarose plugs for 24 hours. The lysis process is repeated once again in the same way. Thereafter, the agarose plugs are washed twice or three times with deionized water. The plugs are then put into 50 ml TE ₅₀ buffer (10 mM Tris-Cl, 50 mM EDTA, pH 8.0) and washed for 12 hours with a roller mix. After the 50 ml TE ₅₀ buffer is replaced with a fresh 50 ml TE ₅₀ buffer, an additional washing is performed on the plugs for 12 hours. Subsequently, the plugs are incubated for 2 hours at 4 Celsius degrees in order to inactivate proteinase K in 0.1 mM Phenylmethylsulfonyl Fluoride (PMSF) buffer, washed with the TE ₅₀ buffer for 24 hours, and put in 0.5 M EDTA to be kept at 4 Celsius degrees.

(3) Pre-electrophoresis of agarose plugs

A dialysis is performed for 3 hours on the agarose plugs placed into 0.5 x TBE buffer (45 mM Tris-base, 1 mM EDTA, 45 mM Boric acid). Then, the agarose plugs are inserted into a preparative slot in 1 % pulsed field certified agarose gel and pulsed field gel electrophoresis (PFGE) is conducted on the inserted agarose plugs by using 0.5 x TBE buffer and a CHEF DR-II apparatus with conditions including a pulse time of 5 seconds for up to 10 hours, a temperature of 12 Celsius degrees and a voltage of 4 V/cm. After this treatment, the plugs are removed from the slot, stored in 50 ml 0.5M EDTA buffer, and dialyzed overnight at 4 Celsius degrees.

(4) Partial digestion of plugs

10 HMW DNA embedded plugs are respectively put into 500 micro liters of the enzyme mixture (1 micro liter of EcoRl [NEB, 2 U/micro liters], 1 micro liter of EcoRl Methylase [NEB, 40 U/micro liter], 25 micro liters of 100 x BSA [10 mg/ml], 5 micro liters of 100 x Polyamine, 50 micro liters of 10 x Methylase buffer, 394 micro liters of DW), and equilibrated for 2 hours at 4 Celsius degrees. Then, the plugs are incubated for 4 hours in a heating block set to 37 Celsius degrees. After the plugs are digested to be treated with 150 micro liters of 0.5 M EDTA, 37.5 micro liters of 20 % N-laurosylascosine and 15 micro liters of proteinase K (20 mg/ml), they are reacted for up to 1 hour at 37 Celsius degrees to inactivate endonuclease. Subsequently, PFGE is conducted with conditions including 1 % pulsed field certified agarose gel, a pulse time between 0.1 and 40 seconds for up to 16 hours and a voltage of 6 V/cm to check the partially digested plugs.

Fig. 2 shows an experimental result in accordance with the present invention, exhibiting HMW DNA embedded agarose plugs of Pieris rapae are checked by using PFGE, wherein the plugs are partially digested by an enzyme mixture following pre-electrophoresis.

Lanes 1 and 7 are shown by PFG lamda marker (NEB) and lanes 2 to 6 depict EcoRl digested DNA molds. EcoRl digestion is performed on one mold with the conditions including 4 micro liters EcoRl (2 U/micro liter), 0.5 micro liter of Methylase (40 U/micro liter), 25 micro liters of 100 X BSA, 5 micro liters of 100 X Polyamine, 50 micro liters of Methylase buffer and 375.5 micro liters of DW.

Referring to Fig. 2, a DNA band including a potential Pieris rapae virus is found at 125 kb, so that designing 5 primers from a nucleotide sequence of a Pieris rapae virus is performed for polymerase chain reaction (PCR) to check whether there is the Pieris rapae virus. The DNA band including the potential Pieris rapae virus is found most vividly when EcoRl 8 U and Methylase 20 U are used after 2 hour pre-electrophoresis.

Fig. 3 shows an experimental result of PCR by using PFGE in accordance with the present invention, wherein PCR is performed on 5 primers designed from the nucleotide sequence of a Pieris rapae virus and the size of the PCR product is the same as the expected size of the nucleotide sequence.

Referring to Fig. 3, the first, second, third, forth and fifth primers have a type of AY-519253-1 (expected size of 227 bp), AY-706575-1 (expected size of 223 bp), AY-428513-1 (expected size of 234 bp), AY-449794-2 (expected size of 212 bp) and AY-519252-1 (expected size of 231 bp), respectively.

The conditions of PCR include heat denaturation (at 96 Celsius degrees for 3 minutes), 35 cycles of PCR (at 96 Celsius degrees for 20 seconds, at 50 Celsius degrees for 20 seconds, and at 72 Celsius degrees for 2 minutes) and last extension (at 72 Celsius degrees for 7 seconds).

Furthermore, 1.6g of the ground samples kept at -80 Celsius degrees is selected and molten in 700 micro liters of a lysis buffer (10 ml of 10 x lysis buffer [0.5 M NaCl, 0.01 M Tris-Cl, 0.01 M EDTA], 42 g of urea, 10 ml of 20 % SDS, and 10 ml of 0.5 M EDTA, pH 8.0). After the molten samples are reacted for up to 15 minutes at 37 Celsius degrees and purified with 700 micro liters of Phenol/Chloroform Isoamylacohol (PCI), genomic DNA of Pieris rapae is acquired and used as a PCR template.

The samples are amplified through PCR from genomic DNA in 10 micro liters of a reaction mixture including 1 micro liter (= 50 ng) of genomic DNA, 0.25 micro liter of polymerase (5 U/micro liter), 0.25 micro liter of dNTP, 0.1 micro liter of 1 % DMSO, 0.5 micro liter of forward primer 5p, 0.5 micro liter of reverse primer 5p, 1 micro liter of 10 X buffer and 6.4 micro liters of DW.

Referring to Fig. 3, it is found that the samples are infected with a Pieris rapae virus as expected.

Accordingly, in order to clarify a sequence of the Pieris rapae virus, only DNA of the Pieris rapae virus is separated from samples used for the experiments of the present invention to construct a fosmid library of the Pieris rapae virus.

If DNA of the Pieris rapae virus is separated from the established samples of Pieris rapae, however, genomic DNA of Pieris rapae is inevitably contaminated. Therefore, after the fosmid library is constructed, the fosmid library may be represented by the 3D pooling strategy and then fosmid clones may be found out by screening the fosmid clones to sequence the found clones. In this connection, it will be described in more detail as follows.

(5) Separation of DNA of Pieris rapae virus from DNA of Pieris rapae

Samples for separating DNA of a Pieris rapae virus from DNA of Pieris rapae are provided by using 15 Pieris rapae DNA embedded agarose molds which were prepared in advance in order to construct the bacterial artificial chromosome (BAC) library of Pieris rapae. For the sake of raising the efficiency of the separation of DNA of a Pieris rapae virus, the above processes of (3) Pre-electrophoresis of agarose plugs and (4) Partial digestion of plugs are repeated in the same manner and then the subsequent processes are proceeded.

DNA of a Pieris rapae virus is separated by PFGE with conditions including an initial pulse time of 0.1 second, a final pulse time of 40 seconds, a temperature of 12 Celsius degrees and a voltage of 6 V/cm, for 14 hours. Furthermore, a lamda ladder PFG marker is used as a size marker to enable just a band of Pieris rapae virus corresponding to 125 kb to be selectively eluted.

After the PFGE treatment is completed, the edge of the gel including a size marker is cut and then put into a staining buffer having ethidium bromide to mark the location of 125 kb with degrees. Next, the portion marked with degrees is cut from the remaining gel and then put into a dialysis bag to collect DNA of the Pieris rapae virus using PFGE with the conditions of 1 % pulsed field certified agarose gel, a pulse time between 0.1 - 40 seconds and a voltage of 6 V/cm, for 14 hours.

Fig. 4 shows an experimental result in accordance with the present invention, indicating the DNA of the Pieris rapae virus corresponding to 125 kb is separated from Pieris rapae DNA embedded agarose molds.

Lanes 1 and 4 are shown by PFG lamda marker (NEB) and lanes 2 and 3 depict EcoRl digested DNA.

Next, it is checked with the eyes whether DNA of the Pieris rapae virus is collected by using PFGE and then the concentration of DNA is determined by using a spectrophotometer (DyNA Quant).

Fig. 5 shows an experimental result in accordance with the present invention, indicating the concentration of DNA that has been collected by PFGE is determined by using a spectrophotometer.

Lanes 1 and 2 show eluted DNA (20 ng loading) and a 1 kb ladder, respectively.

< Fosmid library construction of pieri rapea virus >

End reparing of elutioned Pieris rapae virus DNA is as follows.

Eluted Pieris rapae virus DNA 19 micro liters(=95 nano grams), 10X end repairing buffer 5 micro liters, 2.5mM dNTP 5 micro liters, 10mM ATP 5 micro liters, end repairing enzyme 4 micro liters, at 22 Celsius degrees for 45 minutes. Refining by phenol extraction after reaction, a pCC1FOS vector and Pieris rapae virus DNA are combined to ligate recombined DNA. At this time, the ligation reaction is conducted for 18 hours at 22 Celsius degrees with the conditions including 10 micro liters of DNA (100 ng), 2 micro liters of 10 X ligase buffer, 2 micro liters of 10 mM ATP, 0.4 micro liter of pCC1FOS (500 ng/micro liter), 2 micro liters of ligase and 3.6 micro liters of DW. 25 micro liters of a packaging solution is mixed with 10 micro liters of the ligated product for 90 minutes at 30 Celsius degrees and additionally mixed with 25 micro liters of the packaging solution for 90 minutes at 30 Celsius degrees. Using a dilution buffer, the mixture is finally adjusted to 1 ml and infected in EPI 300 cell (OD600 = 1.05) for 20 minutes at 37 Celsius degrees wherein 20 micro liters of packaged DNA is mixed with 200 micro liters of EPI 300 cell and reacted in total 42 tubes.

The quality of the thus constructed fosmid library is checked by titering one (440 micro liters of a cell stock) of the 42 tubes and screening it (white : blue = 145 : 2). The number of clones is approximately 6000 in total. 96 clones are selected and end sequenced.

(6) Fosmid terminal mapping using BLAST (Basic Local Alignment Search Tool)

Figs. 6 and 7 are experimental results in accordance with the present invention, showing data prior to and posterior to mapping fosmid clones based on the result of blast nt & nr performed on granulovirus and 9 kinds of Pieris rapae, respectively.

For example, Pieris rapae granulovirus and Plutella xylostella granulovirus are shown in Fig. 7.

Referring to Figs. 6 and 7, clones acquired by performing fosmid end sequencing on granulovirus and 9 kinds of Pieris rapae are mapped in order to enable use of minimum clones in a synteny region based on the result of blast nt & nr.

(7) Construction of shot-gun library

Based on the mapped data in the blast nt & nr database, a minimum tailing path is prepared. First of all, 4 fosmid library clones are selected on the basis of the prepared minimum tailing path to measure the size of the clones and construct a shot-gun library.

Fig. 8 shows an experimental result in accordance with the present invention, representing the size of the 4 selected fosmid clone DNAs measured by NotI restriction enzyme digestion of fosmid clone DNAs, and Fig. 9 is a schematic diagram including a flow chart of shot-gun library construction.

Referring to Fig. 8, lanes 2 and 7 are shown by a monocot lamda marker, lane 1 is a 1 kb ladder and lanes 3 to 6 are fosmid Notl digested DNA. Furthermore, the names of the first selected clones are NB-FOS-1-1-F40_A05A02 (lane 5, approximately 30 kb), NB-FOS-1-1-F40_A23B06 (lane 6, approximately 36 kb), NB-FOS-1-1-F40_C07D02 (lane 3, approximately 35 kb) and NB-FOS-1-1-F40_E13E04 (lane 4, approximately 40 kb).

Referring to Fig. 9, a shot-gun library is constructed as follows. First, genomic DNA or BAC DNA isolation and purification are performed. DNA fragments (3 7 kb) are randomly sheared. Small fragments are removed and the size fraction is performed. After Size fraction, DNA is dissolved in 8 micro liters by ethanol precipitation, and the reaction is conducted for 10 min at 37℃ with the condition including 1 micro liters 10X kinase buffer, 1 micro liters kinase enzyme. Refining by phenol extraction after reaction, a pUC118 ready vector and inserted DNA are combined to ligate recombined DNA. At this time, the ligation reaction is conducted for 4 hours at 16 ℃ with the conditions including 10 micro liters of DNA (600 ng), 10 micro liters of ligation mixture (TAKARA, Blunting ligation kit), 1 micro liters of pUC118 ready vector. Then, transformation is performed by electroporation (DH5α). The quality of the thus constructed shot-gun library is checked by titering (40 micro liters of a cell stock, white : blue = 400 : 100). The number of clones is approximately 20,000 in total. 96 clones are selected and sequenced including insert size check, E. coli and vector % check.

(8) 8 X coverage sequencing

The shot-gun library is constructed based on the first selected clones and then plasmid clones which are 8 times larger than each of the selected clones are randomly picked for plasmid preparation, DNA concentration measurement and PCR sequencing. The PCR product is analyzed by using 3730 XL DNA analyzer (applied biosystem).

(9) Finishing

The average read length is approximately 850 bp on a phred score 20 basis. Contig is prepared by performing phredphrap and vector masking on the average read length, and masking, clustering and assembling a repeated sequence. Then, a primer walking is conducted on the prepared contig for a finishing process.

(10) Selection and sequencing of additional clones

The map of the first clone of selected from Pieris rapae granulovirus is constructed and a clone capable of covering 60 k to 85 k is additionally screened. After (8) to (10) processes are additionally performed, the finish process is completed by a single contig.

While the present invention has been shown and described with respect to the preferred embodiments and figures, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and the scope of the present invention as defined in the following claims.