Download A SIMPLE, RAPID MICROASSAY FOR DNA

생명과학기법 보고서 01
A SIMPLE, RAPID
MICROASSAY FOR DNA
Bridget T. HILL and S. WHATLEY
Department of Cellular Pathology, Imperial Cancer Research
Fund, Lincoln’s Inn Fields, London, WC2A 3PX. UK
Received 4 June 1975
생명과학부
20641036 현명재
INDEX
1. Introduction
2. Methods
3. Results and Discussion
Specificity of the mithramycin – DNA microassay
Comparison, using various assay procedures
4. Reference
5. My Opinion
1. Introduction
There is a need for a more sensitive method for assaying DNA
when attempting quantitative biochemical estimations on small
numbers of cells.
Past Method
-Existing techniques for DNA estimations are laborious and often
restricted by their sensitivity.
Lower limit of about 10 pg or approximately l06 cells.
This Method
-Based on the fact that the antibiotic mithramycin binds to
double stranded DNA and fluoresces
As few as 105 cells (or 0.5 pg of pure DNA)
2. Methods
Material
1. Mithracin,
containing mithramycin and mannitol and sodium diphosphate.
2. A stock solution
containing 200ug/ml mithramycin and 300mM MgCl2,
which promotes DNA-mithramycin interaction.
3. Bovine pancreatic Dnase I, yeast RNA type I
4. Duplicate calf thymus DNA standards
containing DNA, mothramycin and MgCl2.
5. The fluorescence of a 1 ml aliquot at 540nm
was determined using an Aminco-Bowmann spectrofluorometer
2. Methods
For investigations of
the relationship between cell number and fluorescence in the presence
of mithramycin,JI cells, a transformed line derived from baby
hamster kidney cells were grown at 37°C in a humidified 5% CO* -95%
air atmosphere using Dulbecco’s medium supplemented with 10% foetal
calf serum.
For estimation of cellular DNA, TA3B cells, a mouse tumour line, and
human embryo lung fibroblasts were grown using Dulbecco’s and
Waymouth’s medium respectively, supplemented with 10% newborn calf
serum.
2. Methods
After suspension of monolayer cultures
The cells were centrifuged
Resuspended in PBS (phosphate buffered saline)
Counted in a haemocytometer
Duplicate aliquots of cell suspension were suspended in PBS to a final
concentration of 10 pg/ml mithramycin and 15 mM MgC 12 .
The fluorescence at 540 nm was determined as above, after sonication
of the samples using an MSE ultrasonicator.
3. Results and Discussion
Fig.1 shows that
the mithramycin assay for DNA is linear,
sensitive and reproducible for as little as
0.5ug/ml of DNA.
The specificity of interaction between
mithramycin and cellular DNA is supported
by the lack of fluorescence in
(i)
(ii)
(iii)
(iv)
deoxyribonuclease-treated DNA,
Acid hydrolysed DNA,
RNA, and
protein, and reduced fluorescence with
heat-denatured DNA (see table l), in
agreement with earlier findings.
3. Results and Discussion
This technique was adapted to
determine the DNA content of small
numbers of cells.
(The cells used were mouse TA3B and JI’s, a
transformed line derived from baby hamster kidney
cells, and human embryo lung fibroblasts.)
For the DNA assay, known numbers of
cells were suspended in PBS containing
10 pg/ml mithramycin and 15 mM MgClz
and were disrupted by sonication prior
to estimating the fluorescence of a 1
ml aliquot.
3. Results and Discussion
Fig.2 shows that there is a relationship
between the number of J1 cells and the
fluorescence due to the mithramycinDNA complex. This assay is accurate for
as few as 105 cells/ml.
Between 104- 105 cells there is some
degree of variation, which may be related
to inaccurate cell sampling at these low
numbers.
Recovery of DNA after cell lysis by
freezing and thawing, as opposed to
sonication, provided less reproducible
results.
3. Results and Discussion
the DNA content of TA3B and human embryo lung cells was 7.4-7.8 and
9.0-9.3 pg/cell respectively.
The results for human cells agree well with those reported previously by
other authors using the modified PCA-diphenylamine reaction, the ethidium
bromide technique, a microspectrophotometric method, and estimations of
the DNA phosphorus content.
3. Results and Discussion
This present method offers several advantages
(i) The assay is rapid, providing an answer in minutes and
requiring no overnight incubation as with the diphenylamine
procedure.
(ii) The DNA content of as few as 105 cells can be determined
accurately.
(iii) The assay is simple and multiple samples can be processed
readily.
(iv) Samples can be stroed conveniently as whole cell pellest at
-20°C prior to assay.
3. Results and Discussion
This present method offers several advantages
(v) The assay is directly applicable to cell suspensions or
monolayers.
No time consuming or tedious extraction procedures are
necessary, as is the case with the diphenylamine assay and
the ethidium bromide technique.
(vi) The method is specific for DNA, and since there is no
interference by RNA or protein, treatment with RNase and/or
removal of protein, as required in other methods is
unnecessary.
(vii) Mithramycin, as Mithracin, is readily available and the
method only involves the preparation of two water based
solutions. The use of corrosive acids and inflammable liquids
is avoided.
4. Reference
[ 1 ] Lowry, 0. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem.
193,265-275.
[2] Burton, K. (1956) Biochem. J. 62, 315-322.
[3] Crissman, H. A. and Tobey, R. A. (1974) Science 184,1297-1298.
[4] Ward, D. C., Reich, E. and Goldberg, I. H. (1965) Science 149, 1259-1263.
[5] Behr, W., Honikel, K. and Hartmann, G. (1969) Europ. J. Biochem. 9, 82-92.
[6] Leyva, A. and Kelley, W. N. (1971) Anal. Biochem. 62,173-179.
[ 7] Karsten, U. and Wollenberger, A. (1972) Anal. Biochem. 46, 135-147.
[8] Blackburn, M. J., Andrews, T. M. and Watts, R. W. E. (1973) Anal. Biochem. 51, l-10.
[9] Rudkin, G. T., Hungerford, D. A. and Nowell, P. C. (1964) Science 144, 1229-1232.
[l0] Davidson, J. N., Leslie, I. and White, J. C. (1951) Lancet, i, 1287-1290.
[ 11] CRC Handbook of Biochemistry. Cleveland (1968) (H. A. Sober, ed.) Chemical
Rubber Co.
5. My Opinion
이것은 DNA assay의 Sensitive를 증대시키는 방법에 대해서 기술한 논문
이다. 기존에 연구된 Method와 달리 Double stranded DNA와 fluoresce에
Antibiotic mithramycin을 binding 시키는 방법을 사용한 Method로서 효율
을 극대화 하였다. 이 논문을 읽고 고찰하며 논문에 사용된 방법으로 얻을
수 있는 효과는 Discussion에 소개하였기 때문에 의의 및 향후 응용 분야
방면으로 생각해 보았다.
일단 이 논문 Introduction에 소개 된 것 과 같이 적은 sample로 DNA
assaying 결과의 효율을 높이는 것은 참으로 중요하다고 생각하였다. 인위
적으로 배양된 세포나 유기체에서의 assay은 실제적으로 제한되지 않은
sample의 양이 존재하므로 기존의 방법으로도 가능하겠지만, 현장 증거물
의 제한적일 수 밖에 없는 법의학적인 측면 또는 특정 희귀 생물의 sample
을 이용한 assay에서는 실험의 실패, 성공을 좌우할 수 있기 때문이다.
그리고 Simple, Rapid한 assay는 실험 자제의 효율을 극대화 하여 경제적,
시간적인 측면에서도 큰 도움이 될 것 이라 생각한다.
앞으로는 이와 같이 기초과학에서 새로운 기술의 발견도 중요하지만은
기존 기술의 효율을 극대화 하여 과학의 진보를 촉진하는 연구도 진행되어
야 한다고 생각한다.