Laboratory of Molecular Genetics, KNULaboratory of Molecular Genetics, KNU

Laboratory of Molecular Genetics, KNULaboratory of Molecular Genetics, KNU

Laboratory of Molecular Genetics, KNULaboratory of Molecular Genetics, KNU

Laboratory of Molecular Genetics, KNULaboratory of Molecular Genetics, KNU

Laboratory of Molecular Genetics, KNULaboratory 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, KNULaboratory of Molecular Genetics, KNU

Structure of A-T and G-C base pairsHydrogen-

bonds 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 whitedisks in the

center

Laboratory of Molecular Genetics, KNULaboratory of Molecular Genetics, KNU

DNA strands are anti-parallel

The first proof was providedIn 1961 by measuring the

ratio of different dinucleotides in DNA. The concentration of

5’AG3’ was equal to 5’CT3’ (asexpected from an antiparallelorientation) and not equal to

5’TC3’ (as expected from a a parallel orientation).

DNA sequencing in 1970sconfirmed this conclusion.

5’

3’

3’

5’

Laboratory of Molecular Genetics, KNULaboratory 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, KNULaboratory of Molecular Genetics, KNU

Laboratory of Molecular Genetics, KNULaboratory of Molecular Genetics, KNU

전기영동  

+ -

Laboratory of Molecular Genetics, KNULaboratory of Molecular Genetics, KNU

DNA is negatively charged due to phosphates on its surface. As a result, it moves towards the positive pole.

Size separation of DNA fragments by electrophoresis in agarose gels

Laboratory of Molecular Genetics, KNULaboratory of Molecular Genetics, KNU

전기영동 

Laboratory of Molecular Genetics, KNULaboratory of Molecular Genetics, KNU

전기영동  * Agarose gel 전기영동 시  DNA 의 이동에 영향을 주는 요인들 1) DNA 분자의 크기   느리게 이동한다 . 2) Agarose 의 농도  느리게 이동한다 . 3) DNA 형태 ( 구조 ) 에 따라 이동속도가 다르다 . supercoiled DNA, linear DNA, open circular DNA 의 순으로 빠르게 이동한다 . 4) 부하되는 전압이   이동속도가 빠르다 . 5) 전기장의 방향도 이동속도에 영향을 미친다 . 6) Ethidium bromide 는  DNA 이동속도를  15% 정도 감소시킨다 . 7) 전기영동 완충용액의 성분과 이온강도도 전기영동 속도에 영향을 준다 .

Laboratory of Molecular Genetics, KNULaboratory of Molecular Genetics, KNU

Distance migrated by a DNA fragment in a gel is related to log10 of its size

Laboratory of Molecular Genetics, KNULaboratory of Molecular Genetics, KNU

pCDNA3.1 vector

Plasmid DNA analysis

Laboratory of Molecular Genetics, KNULaboratory of Molecular Genetics, KNU

PCR analysis

MCF7

MB231

MCF7

MB231

# 386

# 411

IgG

Inp

ut

H3K

9me3

H3A

c

SU

V42

0

H4K

20m

e3

DN

MT

1

KA

P-1

Laboratory of Molecular Genetics, KNULaboratory of Molecular Genetics, KNU

Siz

e M

arke

r 2 uL 4 uL 8 uL

SonicationRIPA lysis

- + - + - ++ + + + + +

Genomic DNA analysis

Laboratory of Molecular Genetics, KNULaboratory of Molecular Genetics, KNU

2) DNA 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 

전기영동