Laboratory of Molecular Genetics, KNU
Laboratory of Molecular Genetics, KNU
Laboratory of Molecular Genetics, KNU
Laboratory of Molecular Genetics, KNU
Laboratory of Molecular Genetics, KNU
Structure of the 4 bases found in DNA
Base + sugar is called a nucleoside
Base + sugar + phosphate is called a nucleotide
Laboratory of Molecular Genetics, KNU
Structure of A-T and G-C base pairs
Hydrogenbonds
are shown
as dotted
red lines.
A-T base
pairs have 2
and
G-C base
pairs have 3
H-bonds
H-bonds
are shown
as thin
flat white
disks in the
center
Laboratory of Molecular Genetics, KNU
DNA strands are anti-parallel
5’
3’
3’
5’
The first proof was provided
In 1961 by measuring the
ratio of different dinucleotides
in DNA. The concentration of
5’AG3’ was equal to 5’CT3’ (as
expected from an antiparallel
orientation) and not equal to
5’TC3’ (as expected from a
a parallel orientation).
DNA sequencing in 1970s
confirmed this conclusion.
Laboratory of Molecular Genetics, KNU
Structure of DNA
Watson and Crick
in 1953 proposed
that DNA is a
double helix in
which the 4 bases
are base paired,
Adenine (A) with
Thymine (T)
and Guanine (G)
with Cytosine (C).
Laboratory of Molecular Genetics, KNU
Laboratory of Molecular Genetics, KNU
전기영동
+
-
Laboratory of Molecular Genetics, KNU
Size separation of DNA fragments
by electrophoresis in agarose gels
DNA is negatively charged due to phosphates on its surface.
As a result, it moves towards the positive pole.
Laboratory of Molecular Genetics, KNU
전기영동
Laboratory of Molecular Genetics, KNU
전기영동
* Agarose gel 전기영동 시 DNA의 이동에 영향을 주는 요인
들
1) DNA 분자의 크기 느리게 이동한다.
2) Agarose의 농도 느리게 이동한다.
3) DNA 형태(구조)에 따라 이동속도가 다르다.
supercoiled DNA, linear DNA, open circular DNA의 순
으로 빠르게 이동한다.
4) 부하되는 전압이 이동속도가 빠르다.
5) 전기장의 방향도 이동속도에 영향을 미친다.
6) Ethidium bromide는 DNA 이동속도를 15% 정도 감소시
킨다.
7) 전기영동 완충용액의 성분과 이온강도도 전기영동 속도에
영향을 준다.
Laboratory of Molecular Genetics, KNU
Distance migrated by a DNA fragment
in a gel is related to log10 of its size
Laboratory of Molecular Genetics, KNU
Plasmid DNA analysis
pCDNA3.1 vector
Laboratory of Molecular Genetics, KNU
DNMT1
KAP-1
SUV420
H4K20me3
H3K9me3
H3Ac
IgG
Input
PCR analysis
# 386
MCF7
MB231
# 411
MCF7
MB231
Laboratory of Molecular Genetics, KNU
Size Marker
Genomic DNA analysis
2 uL
+
4 uL
+
+
+
8 uL
+
+
+
+
+
Sonication
RIPA lysis
Laboratory of Molecular Genetics, KNU
전기영동
Amount of agarose
in gel
(%[w/v])
Efficient range of separation of linear
DNA molecules (kb)
0.3
5~60
0.6
1~20
0.7
0.8~10
0.9
0.5~7
1.2
0.4~6
1.5
0.2~3
2.0
0.1~2