(Gel) Electrophoresis

전기 영동: 전기를 이용하여 분자들을 이

동시킨 후 그 이동거리를 확인하여 분자들

의 특성 결정 혹은 분리하는 방법. 물질의

순도나 분자량(크기) 결정

Gel electrophoresis is used to characterize one of the

most basic properties - molecular mass - of both

polynucleotides (DNA, RNA)

and polypeptides (단백질)

Gel electrophoresis can also be used to determine

the purity of these samples

heterogeneity/extent of degradation

subunit composition.

DNA: The most common gel electrophoresis

materials for DNA molecules is

Agarose, starch and acrylamide.

Vertical & Horizontal

http://www.youtube.com/watch?v=vq759wKCCUQ

http://www.youtube.com/watch?v=8RBs0Ghg_48

DNA agarose gels

The electrophoretic migration rate of DNA

through agarose gels is dependent upon

four main parameters:

molecular size

agarose concentration

conformation of the DNA

applied voltage

Mr 1/log (Mw) (이동거리는 log(분자량)의 역수에 비례)

Molecular Mass (Da)

1/log (MW) i.e. relative Mr

100,000 0.20

50,000 0.21

10,000 0.25

5,000 0.27

1,000 0.33

Log(Mr) 1/[gel농도]

Gel % log(1/Gel %)

(i.e. relative Mr)

2.0 3.2

1.5 4.6

1.0 10.0

0.5 100.0

Conformation of the DNA.

different forms (supercoiled, nicked circular, linear DNA) of the same DNA migrate at different rates.

The applied voltage

typical value for running an agarose gel is 5 volts per cm (length of gel).

In both DNA and protein gels, molecular weight markers are run in a lane at one end of the gel

PCR polymerase chain reaction

중합 효소 연쇄 반응

http://www.youtube.com/watch?v=iQsu3Kz9NYo

http://www.youtube.com/watch?v=HMC7c2T8fVk

http://www.youtube.com/watch?v=2KoLnIwoZKU

http://www.youtube.com/watch?v=DkT6XHWne6E

핵산 - 세포 유전물질

deoxyribonucleoic acid (수백만개의 nucleotide 이중가닥)

Nucleotide = C5 + 인산기 + 염기

염기 purine (adenine, guanine), pyrimidine (thymine/uracil, cytosine)

A=T, GC

크기 (염기 숫자 대장균 4.7Mb)

E. coli 약 900genes, 4x106 b.p

• 유전자 발현과정

– 복제(replication): 자신의 복사본 만드는 과정 • 이중가닥 풀림, 복제포크(replication fork)

• 리딩가닥(leading strand), 래깅가닥(lagging strand)

• 복제종결(termination)

– 전사(transcription) • DNA mRNA (tRNA, rRNA)

– 번역(translation) – mRNA protein – 3개 nucleotides 하나의 아미노산 결정

– 유전자 암호, genetic code

• 시작(initiation)

• 신장(elongation)

• 종결(termination)

Polymerase Chain Reaction

one of the most important advances in biotechnology in the last 20 yrs.

Kary Mullis

invented PCR, received 1993 Nobel prize

idea came while driving from San Francisco to Mendocino

now spends much of his time on surfing

received a $10,000 bonus from employer

employer sold rights for $300,000,000

Polymerase Chain Reaction

PCR is an in vitro technique for the amplification

of a region of DNA which lies between two

regions of known sequence.

PCR amplification is achieved by using

oligonucleotide primers.

These are typically short, single stranded

oligonucleotides which are complementary to the

outer regions of known sequence.

Polymerase Chain Reaction

from a mixture of DNA, a specific sequence is duplicated thousands to millions of times.

uses the fact that DNA polymerase requires primers.

DNA polymerase reactions are done in a tube, with primers that control which sequence gets duplicated.

a thermostable DNA polymerase is used.

heat is used to separate the DNA strands, the reaction cycle is repeated until the target sequence is “amplified.”

Thermal cycling parameters

1. Template denaturation

• initial denaturation of template at 95-100 °C.

2. Primer annealing temperature

• Primer annealing temperature is an important parameter in the success of the PCR experiment.

• The annealing temperature is characteristic for each oligonucleotide:

• it is a function of the length and base composition of the primer as well as the ionic strength of the reaction buffer.

3. Primer extension

• Primer extension is usually performed at 72 °C, or the optimum temperature of the DNA polymerase.

• The length of time of the primer extension steps can be increased if the region of DNA to be amplified is long, however, for the majority of PCR experiments an extension time of 2 minutes is

sufficient to get complete extension.

•

중합효소

Thermus aquaticus (Taq)

내열성 세균의 중합효소

효소 활성 70~74℃

PCR 장점

works with very small amounts of DNA (in theory, a single DNA molecule is sufficient)

primers can be designed to amplify any sequences

reaction products can be analyzed by simple gel electrophoresis.

PCR 단점

design of primers requires that sequence is partially known.

difficult to amplify sequences more than a few kb in length

PCR experiments are easily contaminated – as little as one molecule

DNA sequencing

The ultimate analysis of a DNA molecule

Detects all of the differences between DNA sequences

DNA sequence can be used to predict amino acid sequences of polypeptides

Promoters and other requlatory regions can be identified