KR20000025029A - Expansion method of arrayed amplification of restriction enzyme fragments - Google Patents

Expansion method of arrayed amplification of restriction enzyme fragments Download PDF

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KR20000025029A
KR20000025029A KR1019980041903A KR19980041903A KR20000025029A KR 20000025029 A KR20000025029 A KR 20000025029A KR 1019980041903 A KR1019980041903 A KR 1019980041903A KR 19980041903 A KR19980041903 A KR 19980041903A KR 20000025029 A KR20000025029 A KR 20000025029A
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dna
restriction enzyme
amplification
mrna
adapter
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원세연
박한오
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박한오
주식회사 바이오니아
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Abstract

PURPOSE: An expansion method of arrayed amplification of restriction enzyme fragments is provided which expanses amplified total DNA or mRNA of single organism using PCR(polymerase chain reaction). CONSTITUTION: Using one of type IIs or type IIip restriction enzyme which generates nonspecific single stranded base sequence, total DNA or cDNA obtained from mRNA as a template of single organism is cleaved. The cleaved DNA fragments are ligated with adapters of DNA/RNA hybrid hairpin structure as shown in figure 1, and is treated with exonuclease to remove non-ligated DNA fragments. The ligated DNA fragments are treated with NaOH or RNase to remove RNA of the hairpin structure and to produce double stranded DNA. The obtained double stranded DNA is amplified by PCR.

Description

제한효소 절편의 배열화된 증폭의 전개방법Development of sequenced amplification of restriction enzyme fragments

본 발명은 하나의 생물체가 가지는 전체 DNA, 또는 cDNA를 증폭하여 전개하는 방법에 관한 것으로, 순서가 정해진 라이브러리를 사용하지 않고 중합효소연쇄반응만으로 한 생물체의 유전정보를 빠짐없이 전개할 수 있는 방법에 관한 것이다.The present invention relates to a method for amplifying and developing whole DNA or cDNA of a single organism, and to a method for unfolding genetic information of an organism without polymerase chain reaction without using an ordered library. It is about.

하나의 생물체가 포함하고 있는 DNA와 이로부터 만들어지는 mRNA는 매우 다양하고 미량이므로, 이의 염기서열을 밝히거나 하는 등의 목적에 사용하기 위해서는 예외적인 경우를 제외하고는 모두 증폭과정이 필요하게 된다. 이를 위해 일반적으로 사용되는 두 가지 기술은 첫째로 주로 미생물에 선택적으로 클로닝을 하거나 라이브러리 형태를 만들고, 그 미생물을 다량 증식시킨 후, 원하는 부위를 포함한 것만 대량으로 추출하여 사용한다. 두 번째 방법은 중합효소연쇄반응(PCR)을 이용하여 원하는 부위만을 증폭하는 것이다. 이 때 반드시 원하는 부위의 양쪽 끝 부분의 일정 길이의 염기서열을 미리 알고 있어야 한다는 한계가 있다. 그러나, 이미 염기서열이 완전히 밝혀진 생물체가 아닌 경우에는 일반적으로 생물체가 가진 전체 DNA, 또는 이로부터 만들어지는 전체 mRNA에 대해서 중합효소연쇄반응을 이용하여 빠지는 부분이 없이 증폭하는 것은 불가능하다.DNA contained in one organism and mRNA produced therefrom are very diverse and trace amounts, so amplification is necessary except for exceptional cases in order to reveal its nucleotide sequence. To this end, two commonly used techniques are primarily to selectively clone or microbially form a microorganism, multiply the microorganisms in large quantities, and then extract and use only those containing the desired sites in bulk. The second method is to amplify only the desired site using polymerase chain reaction (PCR). At this time, there is a limitation that the base sequence of the predetermined length of both ends of the desired site must be known in advance. However, if the base sequence is not a fully known organism, it is generally impossible to amplify the entire DNA of the organism or the entire mRNA produced therefrom without using a polymerase chain reaction.

대규모 염기서열 결정 작업을 수행함에 있어서, 한 생물체가 가지는 전체 DNA를 비교적 균일한 크기로 쪼개고, 이것을 증폭시킬 수 있는 운반자에 넣고, 이것의 순서를 정하는 것은 필수적인 과정이다. 이를 위해 일반적으로 사용하는 종래의 대표적인 기술은 순서가 정해진 라이브러리(ordered library)이다. 이 작업은 매우 복잡하고 어려우며, 많은 시간과 비용이 드는 과정이다. 이것은 현시점에 있어서 생물체의 대규모 염기서열 결정 작업에 있어서 가장 큰 장애물 중의 하나로 작용하고 있다.In performing large-scale sequencing, it is essential to break down the whole DNA of an organism into relatively uniform size, put it in a carrier capable of amplifying, and order it. A typical representative technique generally used for this is an ordered library. This task is very complicated and difficult, and is a time consuming and expensive process. This is currently one of the biggest obstacles to the large-scale sequencing of living organisms.

따라서, 본 발명의 목적은 한 생물체가 가지는 전체 DNA, 또는 이로부터 만들어지는 전체 mRNA를 빠지는 부분 없이 증폭하여 전개할 수 있는 방법을 제공함으로써, 그 생물체가 가지는 전체 유전정보에 대한 염기서열결정 등의 목적에 사용할 수 있도록 하는 것이다.Accordingly, an object of the present invention is to provide a method for amplifying and developing the entire DNA of an organism, or the entire mRNA produced therefrom without missing portions, thereby determining the sequencing of the entire genetic information of the organism. It can be used for a purpose.

도 1은 배열에서 행(a) 또는 열(b)을 위해 사용되는 DNA/RNA 혼합 머리핀 구조 어댑터의 하나의 예(여기에서 rN은 리보뉴클레오티드를 뜻하며, 나머지는 일반적인 DNA를 구성하는 각 염기를 뜻한다),1 is an example of a DNA / RNA mixed hairpin structure adapter used for row (a) or column (b) in an array, where rN refers to ribonucleotides, and the other refers to each base constituting common DNA. do),

도 2는 배열에서 행(a) 또는 열(b)을 위해 공통적으로 사용되는 중합효소연쇄반응 및 염기서열 결정 반응용 프라이머의 염기서열의 하나의 예,Figure 2 is an example of the nucleotide sequence of the primer for the polymerase chain reaction and sequencing reaction commonly used for row (a) or column (b) in the arrangement,

도 3은 라이게이션, 엑소뉴클레아제 반응, RNA 분해 반응, 중합효소연쇄반응에 사용되는 16x16 배열의 예(여기에서, AA 등은 각 셀에 사용되는 DNA/RNA 혼합 머리핀 구조의 단일가닥 말단부위의 2개의 염기서열이다)를 나타낸다.Figure 3 is an example of a 16x16 array used for ligation, exonuclease reaction, RNA digestion reaction, polymerase chain reaction (where AA, etc. are single-stranded ends of the DNA / RNA mixed hairpin structure used in each cell It is two base sequences of ().

본 발명은 하나의 생물체가 가지는 전체 DNA, 또는 cDNA를 빠짐없이 증폭하여 전개하는 방법에 관한 것이다.The present invention relates to a method for amplifying and developing whole DNA or cDNA of one organism without exception.

이하 본 발명을 상세히 설명하면 다음과 같다.Hereinafter, the present invention will be described in detail.

본 발명은 다음과 같은 순서의 일련의 과정으로 구성된다. 먼저 한 생물체로부터 전체 DNA, 또는 mRNA를 추출하고, mRNA의 경우에는 cDNA 형태로 만든다.The present invention consists of a series of procedures in the following order. First, whole DNA or mRNA is extracted from an organism, and in the case of mRNA, cDNA forms.

그 다음 상기 DNA를 타입 IIs, 또는 타입 IIip 제한효소 중의 하나로 절단을 한다. 이 때 잘린 조각들의 양쪽 끝에 단일 가닥이 생성되는 제한효소를 사용해야 한다. 이 단일 가닥을 구성하는 염기의 개수는 제한효소에 따라 서로 달라진다.The DNA is then cleaved with either type IIs or type IIip restriction enzymes. You should use restriction enzymes that produce single strands at both ends of the cut pieces. The number of bases constituting this single strand varies with the restriction enzyme.

도 1에서와 같은 형태의 DNA/RNA 혼합 머리핀 구조의 어댑터를 도 3에서와 같은 형태로 구성된 시험관들의 배열에 다음 예와 같은 방식으로 분배한다. 예를 들어, 도 3에서 행이 CG이고 열이 AG인 경우에는, 단일 가닥 말단부위에 CG를 가지는 행을 위한 어댑터와 단일 가닥 말단부위에 AG를 가지는 열을 위한 어댑터를 혼합시켜 놓는다. 여기에서는 단일 가닥을 구성하는 염기의 개수가 2개인 경우를 예로 들었으며. 이 때는 그 시험관의 개수는 256 개로 된다. 만약 단일 가닥을 구성하는 염기의 개수가 위의 예와 다를 경우에는 다른 크기의 배열을 사용해야 하며, 또한 그에 적합한 개수의 단일 가닥을 말단부위에 가지는 어댑터를 사용해야 한다. 단일 가닥 부위의 길이가 N이라면, 배열의 크기는 4N×4N 이 된다. 또한, 말단부위를 형성하는 5' 또는 3'의 방향이 제한효소에 의한 것과 어댑터가 서로 결합이 가능하도록 당연히 동일해야 한다.The adapter of the hairpin structure of the DNA / RNA mixed hairpin structure as shown in FIG. 1 is distributed to the array of test tubes configured as shown in FIG. 3 in the following manner. For example, in FIG. 3 where the row is CG and the column is AG, the adapter for the row with CG at the single stranded end and the adapter for the column with AG at the single stranded end are mixed. In this example, the number of bases constituting a single strand is given as an example. In this case, the number of test tubes is 256. If the number of bases constituting a single strand is different from the above example, an array of a different size should be used, and an adapter having a suitable number of single strands at the end should be used. If the length of a single stranded region is N, then the size of the array 4 N × 4 N Becomes In addition, the direction of 5 'or 3' forming the terminal portion must be naturally the same so that the adapter can be connected to each other by the restriction enzyme.

어댑터를 정해진 대로 시험관들에 분배한 다음에는, 위에서 기술한 제한효소로 처리된 DNA 시료를 모든 시험관들에 대해 적당량 씩 혼합을 한다. 그 다음 라이게이션 반응을 시킨다.After dispensing the adapters into the tubes as defined, the DNA samples treated with the restriction enzymes described above are mixed in appropriate amounts for all tubes. Then let the ligation reaction.

그후, 엑소뉴클레아제로 라이게이션되지 않는 것들을 제거시킨다. 이 때 양쪽 말단부위의 양 가닥의 DNA가 머리핀 형태로 공유결합을 하고 있는 특별한 구조의 DNA 이중나선이 만들어지며, 이 형태로 인해 엑소뉴클레아제의 공격으로부터 보호가 된다.Thereafter, those not ligated with exonuclease are removed. At this time, a double-stranded DNA double helix is formed in which both strands of DNA are covalently bonded in the form of hairpins, which protects against the attack of exonucleases.

그 다음에 NaOH와 같은 알칼리, 또는 RNase로 처리하여 머리핀 구조 부위의 RNA를 분해함으로써 일반적인 형태의 DNA 이중나선으로 만든다. 이렇게 함으로써 DNA의 양쪽 가닥이 분리될 수 있게 되어 중합효소연쇄반응의 수행이 가능해진다.It is then treated with an alkali, such as NaOH, or RNase, to break down the RNA at the hairpin structure to form a common DNA double helix. In this way, both strands of DNA can be separated and the polymerase chain reaction can be performed.

그 다음, 도 2에서 예로든 행을 위한 프라이머와 열을 위한 프라이머를 넣고 중합효소연쇄반응을 수행한다. 이 때, 행을 위한 어댑터와 열을 위한 어댑터는 모두, 프라이머의 3' 말단으로부터 연결된 DNA 절편이 시작되는 부위까지 일정 길이의 추가적인 염기서열을 가지고 있다. 이는 염기서열결정 시에 5' 말단부위가 제대로 읽혀지지 않는 문제를 극복하기 위한 것이며, 추가적으로 어댑터의 머리핀 구조에서 이중나선 부위를 길게 함으로써 더욱 안정화시키기 위한 것이다.Then, put the primer for the row and the primer for the column as shown in Figure 2 to perform a polymerase chain reaction. At this time, both the adapter for the row and the adapter for the column have an additional nucleotide sequence of a certain length from the 3 'end of the primer to the site where the linked DNA fragment starts. This is to overcome the problem that the 5 'terminal portion is not read correctly when sequencing, and is further stabilized by lengthening the double helix in the hairpin structure of the adapter.

중합효소연쇄반응 산물을 폴리아크릴아미드 젤 전기영동 등의 방법으로 분리한다.The polymerase chain reaction product is separated by a method such as polyacrylamide gel electrophoresis.

그 다음 젤 상의 각 밴드에서 DNA를 추출하여 염기서열결정 등에 사용한다.Then, DNA is extracted from each band on the gel and used for sequencing.

상기 기술한 과정을 다른 인식부위를 가지는 제한효소에 대해서 동일하게 수행한다. 서로 다른 두 가지, 또는 그 이상의 제한효소를 사용한 전개로부터 얻어진 염기서열을 서로 중첩시켜서, 시료가 된 생물체가 가지는 전체 DNA의 염기서열을 얻어낼 수 있으며, mRNA의 경우에는 원리적으로 그 생물체가 가지는 모든 종류의 mRNA에 대하여 5'와 3' 말단부위 일부를 제외한 나머지를 알아낼 수 있다. 또한, mRNA의 경우에는 각기 존재하는 양의 차이가 대단히 크기 때문에 발생하는 문제가 있을 수 있는데, 이는 mRNA의 양을 평준화(normalize)시키는 여러 가지 다른 기술을 사용하여 극복해야 한다.The procedure described above is performed in the same way for restriction enzymes having different recognition sites. The nucleotide sequences obtained from the development using two different or more restriction enzymes can be superimposed on each other to obtain the nucleotide sequences of the entire DNA of the sampled organism. In the case of mRNA, in principle, the nucleotide sequences of the organism For all types of mRNA, you can find the rest of the 5 'and 3' ends. In addition, in the case of mRNA, there may be a problem due to the large difference in the amount of each present, which must be overcome by using a number of different techniques to normalize the amount of mRNA (normalize).

본 발명을 사용하여 한 생물체가 가지는 전체 DNA, 또는 전체 mRNA를 빠지는 부분이 없이 중합효소연쇄반응을 이용하여 증폭함으로써, 그 생물체가 가지는 전체 유전정보에 대한 염기서열결정 등의 목적에 사용할 수 있으며, 서로 다른 시료에서 발현되는 mRNA의 정도를 비교하고 측정할 수 있으며 서로 다른 시료의 염기서열에서의 돌연변이 다형상(mutation polymorphism)을 밝힐 수 있다.By using the present invention, the whole DNA or whole mRNA of an organism is amplified using a polymerase chain reaction without any part missing, and thus it can be used for the purpose of sequencing the entire genetic information of the organism. The degree of mRNA expressed in different samples can be compared and measured and the mutation polymorphisms in the nucleotide sequences of different samples can be identified.

Claims (3)

단일 가닥 염기서열을 발생시키는 타입 IIs, 또는 IIip 제한효소중 하나로 어떤 시료의 전체 DNA, 또는 mRNA로부터 만들어진 cDNA를 절단하고,Cleavage of cDNA made from the entire DNA, or mRNA, of a sample with either type IIs or IIip restriction enzymes that generate single stranded sequences, 상기 절단된 절편의 양쪽말단과 여기에 선택적으로 반응하도록 설정된 DNA/RNA 혼합 머리핀 구조의 어댑터를 라이게이션시키고,Ligating the adapter of the DNA / RNA mixed hairpin structure configured to selectively react with both ends of the cleaved fragment, 엑소뉴클레아제를 이용하여 상기 라이게이션 반응에 참여하지 않은 DNA 절편과 어댑터들을 제거하고,Exonucleases are used to remove DNA fragments and adapters that do not participate in the ligation reaction, 상기 라이게이션된 어댑터의 머리핀 구조부분을 이루는 RNA를 알칼리, 또는 RNase를 사용하여 분해하고,RNA constituting the hairpin structure of the ligated adapter is digested using alkali or RNase, 어댑터내의 염기서열에 상보적으로 결합하는 프라이머를 이용하여 중합효소연쇄반응으로 증폭하고,Amplification by polymerase chain reaction using a primer complementarily binding to the base sequence in the adapter, 폴리아크릴아미드 젤 전기영동으로 분리하는 것으로 이루어지는 제한효소 절편의 배열화된 증폭 전개 방법.A method for the sequenced amplification development of restriction enzyme fragments comprising separation by polyacrylamide gel electrophoresis. 제 1 항에 따른 방법의 염기서열결정에서의 사용.Use in sequencing of the method according to claim 1. 제 1 항에 따른 방법의 돌연변이 다형상연구에서의 사용.Use in mutant polymorphism studies of the method according to claim 1.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20040091462A (en) * 2003-04-22 2004-10-28 (주)바이오니아 Quality Control Method for Microorganisms
EP3992995A1 (en) 2020-10-30 2022-05-04 CK Materials Lab Co., Ltd. Magnetorheological fluid and manufacturing method thereof

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4994370A (en) * 1989-01-03 1991-02-19 The United States Of America As Represented By The Department Of Health And Human Services DNA amplification technique
KR920014937A (en) * 1991-01-31 1992-08-25 레이몬드 피. 올뮬러 Amplification method by chain substitution
WO1993001617A1 (en) * 1991-07-08 1993-01-21 Asea Brown Boveri Ab Method for the manufacture of a semiconductor component
WO1993010264A1 (en) * 1991-11-12 1993-05-27 Oncor, Inc. Restriction amplification assay
US5215899A (en) * 1989-11-09 1993-06-01 Miles Inc. Nucleic acid amplification employing ligatable hairpin probe and transcription

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4994370A (en) * 1989-01-03 1991-02-19 The United States Of America As Represented By The Department Of Health And Human Services DNA amplification technique
US5215899A (en) * 1989-11-09 1993-06-01 Miles Inc. Nucleic acid amplification employing ligatable hairpin probe and transcription
KR920014937A (en) * 1991-01-31 1992-08-25 레이몬드 피. 올뮬러 Amplification method by chain substitution
WO1993001617A1 (en) * 1991-07-08 1993-01-21 Asea Brown Boveri Ab Method for the manufacture of a semiconductor component
WO1993010264A1 (en) * 1991-11-12 1993-05-27 Oncor, Inc. Restriction amplification assay

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20040091462A (en) * 2003-04-22 2004-10-28 (주)바이오니아 Quality Control Method for Microorganisms
EP3992995A1 (en) 2020-10-30 2022-05-04 CK Materials Lab Co., Ltd. Magnetorheological fluid and manufacturing method thereof

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