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<Biotechnology techniques>
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특정 유전자 발현 검토
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ㅖㅊ
PCR(polymerase chain reaction)
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RT-PCR: 특정 mRNA
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DNA Microarray(DNA chip): transcriptome
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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
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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
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Source: http://www.bio.davidson.edu/Courses/Bio111/seq.html
Sanger sequencing: chain termination using dideoxynucleotides
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Pyrosequencing
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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
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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 .
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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
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Trasfection
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Transgenic mice
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ㅏㅐ
Knockout mice
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RNAi
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Footprinting(footprint analysis)
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Band shift assay or Electrophoretic mobility shift assay (EMSA)
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Promoter analysisLuciferase/reporter vector
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Receptor binding assay
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Radioimmunoassay (RIA)
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Radioimmunoassay (RIA)