인체 태반 DNA의 정제 및 성상에 관한 연구

Abstract

[영문]

[한글]

DNA추출은 Marmur 및 Doty(1959)법과 hydroxyapatite(HAP) column법 (Markov 및 Ivanov

1974: Meinke등, 1974)등이 주로 이용되고 있으며 인체 DNA에 관한 연구는 그 재료로 백

혈구나 정자등을 이용하여 왔다. Corneo등(1970)은 DNA추출 재료로 인체 태반을 이용하였

으며 Sounders등(1972)은 이의 크기가 전자현미경적 관찰로 약 7×10**9 base pairs(bp)

정도 된다고 보고하였다. Houck등(1978)은 human genome에는 repeated sequence, unique

sequence 및 satellite DNA등이 존재하고 AluI제한효소로만 잘려지는 부위가 있는 300 nu

cleotides repeated sequence, 2000 nucleotide의 unique sequence 및 6∼10%의 satellit

e DNA 도 개재한다고 하며 Deininger 및 Schmid(1976)는 inverted repeated sequence(pal

indromes) 및 repeated sequence의 배열을 관찰 보고하였다.

본 실험에서는 태반을 이용하여 인체 DNA를 추출 정제하고 일부 성상을 밝혀보고자 하

였으며 실험성적을 요약하면 다음과 같다.

1. 인체 태반 DNA의 추출에 있어 HAP법이 시간적으로는 유용하나 대량을 분리하는데는

수정된 Marmur법이 적절하다고 보며, 두 방법으로 추출한 DNA는 모두 257㎚에서 최고 흡

광도치를 나타내었으며 280㎚와의 흡광도비는 1.98로서 높은 순도를 나타내었다.

2. DNA의 온도해리정수(Tm)는 85.7℃였으며 전 DNA의 GC농도는 약 40mole%로 추정되었

다. Cesium chloride밀도차 초원심분리로 주 DNA분획과 상부에 3개의 satellite DNA분획

을 볼 수 있고 UV흡광도(260㎚)에 따라 주 DNA와 satellite DNA S^^1,S^^2를 분리할 수

있었다.

3. 태반 DNA를 단일제한효소로 처리함으로 EcoRⅠ으로 6개, HindⅢ로 1개, BamHⅠ으로

1개, HinfⅠ으로 5개, AluⅠ으로 4개 및 HaeⅢ로 7개의 분획이 출현되었으며 총 15개의

각기 다른 크기의 repeated sequences를 확인할 수 있었고 HPaⅠ로는 어떠한 분획도 나타

나지 않았다.

4. 태반 DNA를 EcoRⅠ처리하여 얻은 repeated sequence중 1819, 1520, 1277 bp에는 Hae

Ⅲ, HinfⅠ 및 AluⅠ에 분해되는 부위가 있었고, 1520bp에는 BamHⅠ부위가, 860과 713bp

내에 HirfⅠ부위가 있었다. 또한 HindⅢ에 의해 생긴 repeated sequence인 1863bp내에는

HaeⅢ, HinfⅠ 및 AluⅠ부위가 있고 BsmHⅠ과 HpaⅠ에 의해 일부만이 분해 (Partial clea

vage)되는 부위도 있었다.

Purification and Characterization of Human Placenta DNA

Jung Hye Kim

Department of Medical Science The Graduate School, Yonsei Uuiuersity

(Directed by Professor Sa Suk Hong, M.D.)

Purification of deoxyribonucleic acid(DNA) ha9 been mainly based on the

traditional method of Marmur and Doty(1959) as well as the method employing

hydroxyapatjte(HAP) column, and leukocytes and sperm were used as a source of human

DNA. However, Corneo et al(1970) have utilized human placenta and reported that the

DNA has 7×10**9 base pairs which consists of repeated sequences and contains

6∼10% of satellite DNA. The repeated sequences are composed of 300 nucleotides and

it is cleaved by the restriction enzyme AluⅠ.

In the present study, human placenta was used as a source of DNA and the nuclear

DNA were purified by both the modified Marmur and Doty procedure and the HAP column

procedure. Furthermore, we have attempted to identify the repeated sequences of

human placenta DNA by using several restriction enzymes.

The results are as fellows:

1. The DNA extraction carried out by employing ice HAP column was lest time

consuming, however, the modified Marmur and Doty procedure was more appropriate for

large scale isolation. The extracted DNA by either method had absorption maximum at

257㎚ and the absorption ratio of 257㎚ vs. 280 ㎚ was 1.98, which indicated high

purity.

2. The melting temperature of purified DNA was 85.7℃ in 1×SSC(0.15M NaCl,

0.015M trisodium citrate, pH 7.0) and the calculated GC content was 40 mole%. By

the cesium chloride density gradient ultracentrifugation, main DNA as well as

satellite DNA S^^1 and S**2 were separated.

3. DNA was digested with a variety of restriction enzymes and subjected to

agarose gelelectrophoresis, 15 different fragment sizes were obtained. Digestion

with EcoRⅠ restriction enzyme produced 6 fragments, HindⅢ; 1, BamHⅠ; 1, HinfⅠ;

5, AluⅠ; 4, and with HaeⅢ; 7. Digestion with Hpa Ⅰ did not show any recognizable

fragment.

4. Additional cleavage sites present within the repeated sequences produced by

initial digestion with EcoRⅠ were identified upon further digestion with HaeⅢ,

HinfⅠ and AluⅠ which were at 1819 base pair(bp), 1520 bp, and 1277 bp,

respectively. Similarly, the cleavage site produced by BamHⅠ was at 1520 bp and

the HinfⅠ sites were at 860 bp and 713 bp.

Within the repeated sequence(1863 bp) produced with HindⅢ digestion, there were

additional cleavage sites which could be further digested with HaeⅢ, HinfⅠ and

AluⅠ, and also partially by BamHⅠ and HpaⅠ.