KR100467666B1 - A Method for Constructing Coding Region Enriched Genomic Library - Google Patents
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- 238000000034 method Methods 0.000 title claims description 7
- 108091026890 Coding region Proteins 0.000 title description 6
- 101710163270 Nuclease Proteins 0.000 claims abstract description 13
- 239000013612 plasmid Substances 0.000 claims description 12
- 239000012634 fragment Substances 0.000 claims description 7
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 3
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 3
- 238000009499 grossing Methods 0.000 claims description 3
- 238000003776 cleavage reaction Methods 0.000 abstract description 7
- 238000004925 denaturation Methods 0.000 abstract description 7
- 230000036425 denaturation Effects 0.000 abstract description 7
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- 235000007164 Oryza sativa Nutrition 0.000 description 15
- 241000209094 Oryza Species 0.000 description 14
- 235000009566 rice Nutrition 0.000 description 14
- 210000004027 cell Anatomy 0.000 description 3
- 210000000349 chromosome Anatomy 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- YLZOPXRUQYQQID-UHFFFAOYSA-N 3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)-1-[4-[2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidin-5-yl]piperazin-1-yl]propan-1-one Chemical compound N1N=NC=2CN(CCC=21)CCC(=O)N1CCN(CC1)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F YLZOPXRUQYQQID-UHFFFAOYSA-N 0.000 description 1
- 241000219195 Arabidopsis thaliana Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241001302160 Escherichia coli str. K-12 substr. DH10B Species 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- 108091060211 Expressed sequence tag Proteins 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 108091092724 Noncoding DNA Proteins 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 210000003763 chloroplast Anatomy 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
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- 210000003470 mitochondria Anatomy 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
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Abstract
본 발명은 S1 뉴클레아제 처리에 의한 유전체 DNA의 부분적인 변성과 절단을 이용한 높은 비율의 암호화 부위를 포함하는 유전체 라이브러리의 제작방법에 관한 것이다. 본 발명에 의하면, S1 뉴클레아제에 의한 부분적인 변성과 그에 의한 절단이 유전체 DNA의 암호화 부위보다는 비암호화 부위에서 보다 효율적으로 수행되므로, 간단하고 용이한 방법으로 높은 비율의 암호화 부위를 포함하는 유전체 라이브러리를 생산할 수 있으며, 이를 통하여 다양한 생물학적 현상의 분석에 크게 기여할 수 있을 것이다.The present invention relates to a method of making a genomic library comprising a high proportion of coding sites using partial denaturation and cleavage of genomic DNA by S1 nuclease treatment. According to the present invention, since partial denaturation and cleavage by S1 nuclease are performed more efficiently at non-coding sites than on coding sites of genomic DNA, genomes containing a high proportion of coding sites in a simple and easy manner. Libraries can be produced, which will greatly contribute to the analysis of various biological phenomena.
Description
본 발명은 높은 비율의 암호화 부위를 포함하는 유전체 라이브러리의 제작방법에 관한 것이다. 좀 더 구체적으로, 본 발명은 S1 뉴클레아제 처리에 의한 유전체 DNA의 부분적인 변성과 절단을 이용한 높은 비율의 암호화 부위를 포함하는 유전체 라이브러리의 제작방법에 관한 것이다.The present invention relates to a method of making a genomic library comprising a high proportion of coding sites. More specifically, the present invention relates to a method of making a genomic library comprising a high proportion of coding sites using partial denaturation and cleavage of genomic DNA by S1 nuclease treatment.
오늘날, 다양한 생물학적 현상의 분석에 유전자가 일반적으로 사용됨에 따라, 고도로 밀집된 DNA를 포함하는 유전자 칩의 중요성이 날로 높아지고 있다. 따라서, 칩 상에 점적되는 DNA의 개수가 중요한 요소로 작용하게 되고, 이를 위해서는 공증된 유전체 서열을 포함하는 라이브러리가 필수적으로 요구된다. 그러나, 식물이나 포유류 등의 고등생물의 유전체는 그 크기가 매우 커서 완전한 서열결정이 어렵고, 서열이 완전하게 결정되었다 하더라도 암호화 부위 외에 불필요한 삽입 부위와 인트론 부위가 전체의 적어도 50% 이상을 차지하고 있기 때문에, 그들의 라이브러리 역시 비암호화 부분의 비중이 매우 높고, 상업화되어 상용되고 있는 DNA 라이브러리 역시 프라이머 제작을 위한 ORF의 확인을 위해서나 또는 PCR을 위한 주형으로서만 사용할 수 있을 뿐이어서 효용성이 높지 않았다.Today, as genes are commonly used in the analysis of various biological phenomena, the importance of gene chips containing highly dense DNA is increasing day by day. Therefore, the number of DNA deposited on the chip is an important factor, which requires a library containing notarized genomic sequences. However, since genomes of higher organisms such as plants and mammals are so large that complete sequencing is difficult, even if the sequence is completely determined, unnecessary insertion sites and intron sites occupy at least 50% or more of the entire region. In addition, their libraries have a high proportion of non-coding portions, and commercially available DNA libraries are not only highly useful because they can be used only for the identification of ORF for primer preparation or as a template for PCR.
따라서, 높은 비율의 암호화 부위를 포함하는 유전체 라이브러리를 제작할 수 있는 기술을 개발하여야 할 필요성이 끊임없이 대두되었다.Thus, there is a constant need to develop a technique for producing a genomic library containing a high proportion of coding sites.
이에, 본 발명자들은 높은 비율의 암호화 부위를 포함하는 유전체 라이브러리를 제작하기 위한 방법을 개발하고자 예의 연구노력한 결과, 유전체 DNA를 S1 뉴클레아제로 처리하여 부분적으로 변성시키고 절단시킨 다음, 절단된 DNA 단편의 양 말단을 DNA 폴리머라제를 이용하여 평활화시키고, 이를 플라스미드에 삽입하여 제작한 라이브러리는 암호화 부위를 높은 비율로 포함하여 낮은 중복도 값을 나타내는 것을 확인하고, 본 발명을 완성하게 되었다.Accordingly, the present inventors have made intensive efforts to develop a method for manufacturing a genomic library including a high proportion of coding sites. As a result, the present inventors have treated the genomic DNA with S1 nuclease, partially denatured and cleaved, and then, Both ends were smoothed using DNA polymerase, and the library prepared by inserting the same into the plasmid was confirmed to exhibit a low redundancy value by including a high proportion of coding sites, thereby completing the present invention.
결국, 본 발명의 주된 목적은 높은 비율의 암호화 부위를 포함하는 유전체라이브러리의 제작방법을 제공하는 것이다.After all, the main object of the present invention is to provide a method for fabricating a genome library comprising a high proportion of coding sites.
도 1은 본 발명의 유전체 라이브러리의 제작방법을 나타내는 간단한 모식도이다.1 is a simple schematic diagram showing a method for producing a genome library of the present invention.
도 2a 및 도 2b는 아라비돕시스 및 벼 라이브러리의 클론의 중복성을 나타내는 그래프이다.2A and 2B are graphs showing the redundancy of clones of Arabidopsis and rice libraries.
본 발명의 높은 비율의 암호화 부위를 포함하는 유전체 라이브러리의 제작방법은 (i) 유전체 DNA를 S1 뉴클레아제로 처리하여 부분적으로 변성시키는 단계; (ii) 전기 부분적으로 변성된 유전체 DNA를 추가의 S1 뉴클레아제로 처리하여 절단하는 단계; (iii) 절단된 유전체 DNA 단편의 양 말단을 평활화시키는 단계; 및, (iv) 양 말단이 평활화된 유전체 DNA 단편을 플라스미드에 삽입시켜, 라이브러리를 제작하는 단계를 포함한다. 본 발명에 의하면, S1 뉴클레아제에 의한 부분적인 변성과 그에 의한 절단이 유전체 DNA의 암호화 부위보다는 비암호화 부위에서 보다 효율적으로 수행되므로, 간단하고 용이한 방법으로 높은 비율의 암호화 부위를 포함하는 유전체 라이브러리를 생산할 수 있으며, 이를 통하여 다양한 생물학적 현상의 분석에 크게 기여할 수 있을 것이다.A method of making a genomic library comprising a high proportion of coding sites of the invention comprises the steps of: (i) treating the genomic DNA with S1 nucleases to partially denature it; (ii) treating said partially denatured genomic DNA with additional S1 nuclease to cleave; (iii) smoothing both ends of the cleaved genomic DNA fragment; And (iv) inserting genomic DNA fragments blunted at both ends into the plasmid to produce a library. According to the present invention, since partial denaturation and cleavage by S1 nuclease are performed more efficiently at non-coding sites than on coding sites of genomic DNA, genomes containing a high proportion of coding sites in a simple and easy manner. Libraries can be produced, which will greatly contribute to the analysis of various biological phenomena.
이하, 실시예를 통하여 본 발명을 보다 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 구체적으로 설명하기 위한 것으로, 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.
실시예 1: 유전체 DNA 라이브러리 제작 Example 1 Genomic DNA Library Construction
벼와 아라비돕시스의 유전체 DNA 라이브러리를 제작하였다(참조: 도 1): 벼(Oryza sativacv. Nipponbare) 및 아라비돕시스 살리아나(Arabidopsis thaliana)로부터 유전체 DNA를 분리하고(참조: Shure M.et al., Cell, 35:255-233, 1993), 2㎍의 유전체 DNA를 S1 뉴클레아제(150 units, TAKARA, Japan)를 포함하는 50㎕의 완충용액에 용해시킨 다음, 70℃에서 5 내지 10분 동안 배양하여 전기 유전체 DNA를 부분적으로 변성시키고, 여기에 S1 뉴클레아제(150 unit)를 포함하는 완충용액을 추가로 첨가하여 5 내지 10분 동안 배양하였다. 배양 후, DNA를 전기 용융점이 낮은 아가로스 겔(low melting agarose gel)에서 전기영동하여, 0.7 내지 1.5kb 크기의 DNA 단편만을 추출하여 수득하였다. 전기 수득된 DNA의 양 말단을 dNTP와 T4 DNA 폴리머라제를 이용하여 평활화시키고, SmaI으로 절단되고 CIP로 탈인산화된 pBluescript 플라스미드에 삽입하여, 벼와 아라비돕시스를 위한 재조합 플라스미드 각 722개와 1005개를 포함하는 라이브러리를 제작하였다. 그런 다음, 전기 라이브러리의 각 재조합 플라스미드를 컴피턴트 세포인E. coliDH10B(Gibco BRL, USA)에 도입하여 세포를 형질전환시키고, 암피실린을 포함하는 고체배지에 도말하여 배양하였다. 전기 배지에 콜로니 형태로 나타난 형질전환된 클론을 취하여 재조합 플라스미드 DNA를 분리하고, ABI 3700 DNA 서열분석기(Perkin Elmer, USA)를 이용하여 각 플라스미드의 염기서열을 결정하였다.A genomic DNA library of rice and Arabidopsis was prepared (FIG. 1): genomic DNA was isolated from rice ( Oryza sativa cv. Nipponbare) and Arabidopsis thaliana (Shure M. et al ., Cell) , 35: 255-233, 1993), 2 μg of genomic DNA was dissolved in 50 μl of buffer containing S1 nuclease (150 units, TAKARA, Japan), and then incubated at 70 ° C. for 5 to 10 minutes. By partially denaturing the electrical genomic DNA, an additional buffer solution containing S1 nuclease (150 unit) was added thereto and incubated for 5 to 10 minutes. After incubation, the DNA was electrophoresed on a low melting agarose gel to obtain a DNA fragment of 0.7 to 1.5 kb in size. Both ends of the previously obtained DNA were blunted using dNTP and T4 DNA polymerase, inserted into pBluescript plasmid digested with SmaI and dephosphorylated with CIP, containing 722 and 1005 recombinant plasmids each for rice and arabidopsis. A library was produced. Then, each recombinant plasmid of the electrical library was introduced into competent cells E. coli DH10B (Gibco BRL, USA) to transform the cells, and plated and cultured in solid medium containing ampicillin. Recombinant plasmid DNA was isolated from the transformed clones in colony form in the medium, and the nucleotide sequence of each plasmid was determined using an ABI 3700 DNA sequencer (Perkin Elmer, USA).
실시예 2: 라이브러리의 서열분석 Example 2 Sequencing of Libraries
실시예 2-1: 아라비돕시스 라이브러리의 분류 Example 2-1 Classification of Arabidopsis Libraries
상기 실시예 1에서 결정된 아라비돕시스 라이브러리의 각 재조합 플라스미드가 포함하는 유전체 DNA 단편 서열을 공증된 유전체 서열과 비교하여, 암호화 서열과 비암호화 서열로 분류하였다: 상기 결정된 아라비돕시스 라이브러리의 각 재조합 플라스미드 염기서열에서 플라스미드 서열을 제거하고, 20 이상의 QV(quality value) 값을 가지며 200bp 이상의 크기를 갖는 서열만을 BALST 알고리즘으로 분석하여 BALST 데이터베이스의 공증된 아라비돕시스 유전체 서열과의 상동성을 검사하였다. 그런 다음, 공증된 아라비돕시스 유전체 서열과 90% 이상의 상동성을 갖는 서열 및 공증된 유전체 서열의 암호화 부위와 90개 이상의 뉴클레오티드가 일치한 서열만을 선택하여 암호화 서열로 분류하였는데, 암호화 서열로의 분류를 위한 최소한의 길이를 90bp로 결정한 것은A. thaliana, 벼 및 인간의 대부분의 엑손은 90bp 이상의 크기를 갖기 때문이다.The genomic DNA fragment sequences contained in each recombinant plasmid of the Arabidopsis library determined in Example 1 were classified into coding sequences and non-coding sequences: plasmids in each recombinant plasmid sequence of the Arabidopsis library determined above. Sequences were removed, and only sequences having a quality value (QV) value of 20 or greater and a size of 200 bp or greater were analyzed by the BALST algorithm to examine homology with the notified Arabidopsis genome sequence of the BALST database. Then, only those sequences having at least 90% homology with the notarized Arabidopsis genome sequence and sequences having at least 90 nucleotides identical to the coding region of the notarized genome sequence were selected and classified as coding sequences. The minimum length of 90 bp was determined because A. thaliana , rice and most of exons in humans have a size of 90 bp or more.
실시예 2-2: 벼 라이브러리의 분류 Example 2-2 : Classification of Rice Library
GenBank의 nr(non-redundant) 데이터베이스와 EST 라이브러리, 그리고 공증된 벼의 BAC 라이브러리를 사용한다는 것을 제외하고는 전기 실시예 2-1과 동일한 방법을 사용하여, 벼 라이브러리의 각 재조합 플라스미드가 포함하는 유전체 DNA단편 서열을 공증된 유전체 서열과 비교하여, 암호화 서열과 비암호화 서열로 분류하였다.The genome contained in each recombinant plasmid of the rice library, using the same method as in Example 2-1, except that GenBank's nr (non-redundant) database, EST library, and notarized rice BAC library were used. DNA fragment sequences were compared to notarized genomic sequences and classified into coding and noncoding sequences.
실시예 2-3: 분류된 서열의 분석 Example 2-3 Analysis of Sorted Sequences
전기 실시예 2-1 및 2-2에서 암호화 서열로 분류된 서열을 분석하고, 상기 실시예 1에서 제작한 라이브러리 중 비암호화 부위가 차지하는 비율을 라이브러리의 중복도(redundancy)로 계산하였다. 도 2a 및 도 2b는 아라비돕시스 및 벼 라이브러리의 클론의 중복성을 나타내는 그래프인데, 도 2a 및 도 2b에서 보듯이, 대부분의 클론들이 서로 중복되지 않은 서열을 포함하는 것을 확인할 수 있었고, 이를 통하여 계산된 라이브러리의 중복도는 아라비돕시스와 벼 모두에서 1.1 보다 낮은 값으로 나타났다.Sequences classified as coding sequences in Examples 2-1 and 2-2 were analyzed, and the proportion of non-coding sites in the library prepared in Example 1 was calculated as the redundancy of the library. 2A and 2B are graphs showing the redundancy of clones of Arabidopsis and rice libraries. As shown in FIGS. 2A and 2B, it was confirmed that most of the clones contained sequences that did not overlap each other, and the library was calculated therefrom. The redundancy of was lower than 1.1 in both Arabidopsis and rice.
또한, 전기 암호화 서열의 BLAST 분석 결과, 라이브러리에서 암호화 서열로 분류된 서열은 각각 염색체, 엽록체 및 미토콘드리아의 DNA와 일치하였는데, 전체 722개의 아라비돕시스 라이브러리의 경우, 각각 604개, 104개, 14개이었고, 전체 1005개의 벼 라이브러리의 경우, 각각 970개, 33개, 2개이었으며, 전기 각 서열은 공증된 아라비돕시스 라이브러리 또는 공증된 BAC 라이브러리의 서열과 서열 및 크기가 90% 이상 일치하였고, 이로써, 라이브러리의 염색체 DNA 중 엑손을 포함하는 서열의 비율은 아라비돕시스와 벼에서 각각 42%와 45.5%로 계산되었다.In addition, as a result of BLAST analysis of the electrocoding sequence, the sequence classified as the coding sequence in the library was consistent with the DNA of chromosomes, chloroplasts and mitochondria, respectively, which was 604, 104 and 14 for the total of 722 arabidopsis libraries, For the total 1005 rice libraries, there were 970, 33, and 2, respectively, and the sequence and size of each of the foregoing sequences coincide with the sequence of notarized Arabidopsis library or notarized BAC library by 90% or more, thereby chromosomes of the library The proportion of exon-containing sequences in the DNA was calculated to be 42% and 45.5% in Arabidopsis and rice, respectively.
한편, 아라비돕시스 유전체에서 암호화 부위는 약 25%인 것으로 알려져 있고, 벼의 경우는 정확하게 밝혀진 바는 없지만, 일부의 보고에 따라 약 20% 미만일 것으로 예상되고 있다. 따라서, 상기 실시예 1에서 제작한 아라비돕시스 및 벼의 라이브러리는 각각 약 70%(42/25 = 1.68)와 82%(45.5/20 = 1.82) 이상의 암호화 부위를 포함하는 것으로 계산되었다.On the other hand, in the Arabidopsis genome, the coding region is known to be about 25%, the case of rice is not exactly known, but some reports are expected to be less than about 20%. Therefore, the libraries of Arabidopsis and rice prepared in Example 1 were calculated to contain at least about 70% (42/25 = 1.68) and 82% (45.5 / 20 = 1.82) coding sites, respectively.
이상의 결과에 의하여, S1 뉴클레아제에 의한 부분적인 변성과 그에 의한 절단은 유전체 DNA의 암호화 부위보다는 비암호화 부위에서 보다 효율적으로 수행되며, 이를 암호화 부위를 높은 비율로 포함하는 유전체 라이브러리의 제작에 이용할 수 있음을 확인하였다.As a result, partial denaturation and cleavage by S1 nuclease are performed more efficiently at the non-coding region than at the coding region of the genomic DNA, which can be used to construct a genomic library containing a high proportion of the coding region. Confirmed that it can.
이상에서 상세히 설명하고 입증하였듯이, 본 발명은 S1 뉴클레아제 처리에 의한 유전체 DNA의 부분적인 변성과 절단을 이용한 높은 비율의 암호화 부위를 포함하는 유전체 라이브러리의 제작방법을 제공한다. 본 발명에 의하면, S1 뉴클레아제에 의한 부분적인 변성과 그에 의한 절단이 유전체 DNA의 암호화 부위보다는 비암호화 부위에서 보다 효율적으로 수행되므로, 간단하고 용이한 방법으로 높은 비율의 암호화 부위를 포함하는 유전체 라이브러리를 생산할 수 있으며, 이를 통하여 다양한 생물학적 현상의 분석에 크게 기여할 수 있을 것이다.As described and demonstrated in detail above, the present invention provides a method for producing a genomic library comprising a high proportion of coding sites using partial denaturation and cleavage of genomic DNA by S1 nuclease treatment. According to the present invention, since partial denaturation and cleavage by S1 nuclease are performed more efficiently at non-coding sites than on coding sites of genomic DNA, genomes containing a high proportion of coding sites in a simple and easy manner. Libraries can be produced, which will greatly contribute to the analysis of various biological phenomena.
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| p. Biochem. Biotechnol., Vol.29(1), pp.91-100 (1999.02.) * |
| Preparation of a genomic library using a TA vector. Prep Biochem Biotechnol. 1999 Feb;29(1):91-100. * |
| Rapid and efficient method for cloning of blunt-ended DNA fragments. Gene. 1987;51(1):69-75. * |
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