KR910004119B1 - Individual conform method using dna - Google Patents

Individual conform method using dna Download PDF

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KR910004119B1
KR910004119B1 KR1019880011426A KR880011426A KR910004119B1 KR 910004119 B1 KR910004119 B1 KR 910004119B1 KR 1019880011426 A KR1019880011426 A KR 1019880011426A KR 880011426 A KR880011426 A KR 880011426A KR 910004119 B1 KR910004119 B1 KR 910004119B1
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dna
individual
molecules
membrane
probe
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KR900005176A (en
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하지홍
박용복
장형수
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하지홍
박용복
장형수
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing

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Abstract

A DNA is purifyed from the body fluid of a higher animal, decomposed by treating single- or multi-restricition enzyme, separated by using electrophoresis, and transfered onto a membrane. The otained DNA molecule is mixed with a labelled DNA probe as a standard, washed with SCC soln., and detected by autoradiography.

Description

디옥시 핵산(DNA)분자를 이용한 개체 확인방법Identifying individuals using deoxynucleic acid (DNA) molecules

본 발명은 디옥시 핵산(이하 DNA로 약칭함*) 분자의 특성을 이용하여 친자감별, 범죄감식 및 유전병의 검색 등과 같은 개체확인이 가능토록한 것이다.The present invention enables the identification of individuals such as paternity, crime, and genetic diseases by using the properties of deoxy nucleic acid (hereinafter abbreviated as DNA) molecules.

종래에는 개체확인을 위해 주로 지문을 이용하는 것이 대종으로 행해졌으나, 보다 정밀하고 확실한 개체 확인을 위해서는 보다 새로운 방법이 요망되는 것이 현실이다.Conventionally, the use of fingerprints mainly for identification is largely performed, but in reality, a new method is required for more precise and reliable identification of individuals.

고등동물의 염색체에는 repeat DNA 염기 서열이 있는데 동일 종류내의 개체에 따라서 염기서열이 모두 다른것으로 밝혀진다. repeat DNA를 이용하여 개체확인을 한다면 오차가 없는 정확한 개체확인이 가능하기 때문에 범죄감식은 물론 친자감별 등에 광범위하게 그리고 유효적절하게 활용할 수 있는 것이므로 그 구체적 내용을 상세히 설명하면 다음과 같다.The higher animal chromosome has a repeat DNA sequence, which is found to be different for each individual within the same species. If you use the repeat DNA to identify the individual, it is possible to identify the exact individual without error, so that it can be widely and effectively used for crime recognition as well as paternity discrimination.

(1) 고등동물(사람 포함)의 체액으로부터 DNA를 분리정제한다. 이때 정제방법은,(1) DNA is separated and purified from the body fluids of higher animals (including humans). At this time, the purification method,

첫째 : 혈액의 경우 용해완충액을 가해 균질화(hemogenize)한후 원심분리하여 핵을 모우고 여기에 proteinase K를 처리하고 phenol, chloroform을 여러차례 가해 단백질을 제거한후 70% ethanol에 침전시켜 말린 다음 TE완충액에 녹여 사용한다.First: In the case of blood, homogenize (hemogenize) by adding lysis buffer, centrifuge to collect nuclei, and then process proteinase K, add phenol and chloroform several times to remove proteins, settle in 70% ethanol, dry and dissolve in TE buffer. do.

둘째 : 정액이나 머리카락 뿌리부분으로부터 DNA을 분리해야할 경우에는 공지된 PCR(polymerase chain reaction)법을 도입해서 증폭시킨다.Second: When DNA is to be separated from semen or hair roots, amplification is carried out by introducing a known PCR (polymerase chain reaction) method.

(2) 정제한 DNA를 단일 혹은 다중제한 효소로 분해한후 전기영동장치를 이용하여 agarose gel상에서 전개시키고(이유 : 결합(hybridization)을 위한것임) 전개 분리된 DNA를 적절한 막(menbrane)으로 옮긴다.(2) The purified DNA is digested with single or multiple restriction enzymes and then developed on an agarose gel using electrophoresis (reason: for hybridization). The developed DNA is transferred to an appropriate membrane. .

(3) 막(membrane)상의 DNA와 적절하게 결합할 수 있는 시료(DNA probe)를 제조하기 위하여 다음과정을 거친다.(3) Proceed as follows to prepare DNA probe which can bind with DNA on membrane properly.

즉, 특정동물의 DNA로부터 소위성(mini Satellites) 염기로막을 순수분리한후 대장균 균주내에서 PBR322를 이용하여 분자복제(cloming)한다. 필요에 따라 소위성 염기토막을 생산한후 표식(Labelling)을 하여 시료(DNA probe)로 사용한다.In other words, the membrane is purified from the DNA of a specific animal with so-called (mini Satellites) base and then molecularly cloned using P BR 322 in E. coli strains. If necessary, so-called basic soil membrane is produced and labeled (Labelling) to use as a sample (DNA probe).

(4) 막(menbrane)상의 DNA와 Lebelling된 DNA probe을 적절한 조건에서 혼합한후 SSC용액(시약)으로 3회이상 씻은후 방사선 자동촬영(autoradiograph)을 하여 육안으로 확인할 수 있게한다.(4) After mixing the DNA on the membrane and the labeled DNA probe under appropriate conditions, wash it three times or more with SSC solution (reagent), and then perform radioradiographing for visual confirmation.

(5) Autoradiograph결과가 각 개체에 대한 DNA지문이다.(5) Autoradiograph results are DNA fingerprints for each individual.

이상과 같은 방법으로 만들어진 DNA개체확인방법(지문법)은 각 개체간에서 관찰되는 유전자 염기서열의 다양성에 근거한 분석방법이며, 지구상 50억인구의 각 개체는 서로간에 완전히 다른 독특한 유전자 염기서열을 가지고 있을뿐아니라 일단 부모로부터 공여받은 유전자 염기서열은 외부적인 환경변화에는 무관하게 일생을 통해 거의 불변인 상태로 유지되는 까닭에 언제라도 확인이 가능한 개체판별 효과가 있을 뿐아니라 본 DNA지문결과가 갯수와 간격이 서로다른 10개-20개 막대로 나타나기 때문에 기존의 손가락 지문보다 확인대조가 쉽고 computer를 이용한 자동조사 방법확립에 대단히 유리하며 다른 개체에서 갯수와 간격이 동일한 지문이 나올 확률이 전무(5×10-19)하여 과학적이고 신뢰성이 높은 잇점이 있다.The DNA identification method (fingerprinting method) made by the above method is an analysis method based on the diversity of the gene sequences observed between each individual, and each individual of 5 billion population on earth has a unique gene sequence that is completely different from each other. In addition, the gene sequence received from the parents is almost invariant throughout life regardless of external environmental changes, so there is an individual discrimination effect that can be identified at any time. Since it is represented by 10 to 20 different bars, it is easier to check and compare with the existing finger prints, and is very advantageous for establishing an automatic investigation method using a computer. -19 ), which has the advantage of being scientific and reliable.

Claims (3)

고등동물의 체액에서 분리정제한 개체의 DNA분자와 기준이 되는 표식(Labelling) DNA probe분자를 결합한후 이를 확인하는 감광필름을 만들어 이용케한 디옥시헥산 분자를 이용한 개체확인방법.Individual identification method using a deoxyhexane molecule made by using a photosensitive film that combines the DNA molecules of the individual purified from the body fluids of higher animals and the labeling DNA probe molecules. 상기 1항에서 기준이되는 DNA probe은 특정동물의 DNA중에서 Satellite 혹은 repeat DNA를 순수 분리하여 유전공학적인 방법에 의해 cloning한후 대량생산하여 Labelling하는 것.The DNA probe, which is the reference in the above 1, is to separate the satellite or repeat DNA from the DNA of a specific animal, clone it by genetic engineering method, and then mass produce and label. 상기 1항에서 개체의 DNA분자와 Labelling된 DNA probe결합확인을 위해 방사성 동위원소나 형광물질을 이용하여 방사선이나 형광에 의한 자동 감광작용으로 현상이 되게한것.In order to confirm the binding of labeled DNA probes to individual DNA molecules in the item 1, the phenomenon is caused by automatic photosensitive action by radiation or fluorescence using radioactive isotopes or fluorescent substances.
KR1019880011426A 1988-09-03 1988-09-03 Individual conform method using dna KR910004119B1 (en)

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KR910004119B1 true KR910004119B1 (en) 1991-06-22

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