<Biotechnology techniques>

특정 유전자 발현 검토

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PCR(polymerase chain reaction)

RT-PCR: 특정 mRNA

DNA Microarray(DNA chip): transcriptome

Applications of NGS

NGS

• Whole genome (re)sequencing

• de novo genome assembly (much harder with shorter reads)

• Variant detection (SNPs, indels) and copy number, structural variation

• Targeted resequencing

• Exon sequencing

• Trancriptome

• Whole transcriptome sequencing

• Gene expression profiling

• Small RNA sequencing

• Epigenome

• Chromatin Immunoprecipitation sequencing (Chip-Seq)

• Methylation analysis

-MeDIP(-MeDIP(Methylated DNA Immunoprecipitation ):Anti m5C specific antibody 를 이용한 pull downUnknown C methylation 영역 찾기에 유리

Next Generation Sequencing

Next Generation Sequencer: Three Leading Platforms

454 GS FLX Titanium (Roche)

5500xl SOLiD(AB)

HiSeq2000(Illumina)

Bp per run 1Gb(7Gb/7day)

300Gb 150-200 Gb(540-600)

Read length Av. 400 bp 35-75 bp 50-100 bp

Run time 10 hr 1-7 days 4-8 days

Sample ? 2 genome 2 genomeWith 3730s capillary, ~60 Mb per year

Source: http://www.bio.davidson.edu/Courses/Bio111/seq.html

Sanger sequencing: chain termination using dideoxynucleotides

Pyrosequencing

Kim-Chulhong, PBBRC 12

• 2-DE by subfractionation methods

pH acidic basic

M

W

Kidney tissue, homogenated post-mitochondrial protein, TCA/acetone, colloidal coomassie blue staining

2 D electrophoresis/MALDI-TOF MASS: proteome 분석

Kim-Chulhong, PBBRC 13

Advantage of MALDI-TOF MS1. 빠르다 . 즉 이온화 및 분석물의

검출이 모두 밀리세컨드안에 끝난다 .2. 결과가 분명하다 . 왜냐하면

근본적으로 질량 대 전하비 (mass-to-charge ratio:m/z) 에 근거하기 때문에 핵산의 2 차 구조에 영향을 받는 전기영동이나 hybridization-array (e.g. DNA chip) 보다 정확하다 .

3. 모든 과정 , 시료의 준비에서 결과 처리까지의 과정이 완벽하게 자동화 가능하다 . 따라서 MALDI-TOF MS는 시료의 대량분석 (high-throughput) 의 가능성을 제공한다 .

Kim-Chulhong, PBBRC 14

MALDI-TOF MS• Reflector Mode : isotope peak 분리가능

– Ion mirror (reflector) 를 사용하여 비행거리를 증가시킴으로 resolution 을 높인다

• Linear Mode : resolution 이 떨어진다 .

Flight TubeDetector

Ion Source

4-25 kV

Reflector (Ion Mirror)

Liner mode

R L

Trasfection

Transgenic mice

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Knockout mice

RNAi

Footprinting(footprint analysis)

Band shift assay or Electrophoretic mobility shift assay (EMSA)

Promoter analysisLuciferase/reporter vector

Receptor binding assay

Radioimmunoassay (RIA)

Radioimmunoassay (RIA)