1. <Biotechnology techniques>

4. 특정 유전자 발현 검토

5. PCR(polymerase chain reaction)ㅖㅊ

6. RT-PCR: 특정 mRNA

7. DNA Microarray(DNA chip): transcriptome

8. Applications of NGS Next Generation Sequencing
Whole genome (re)sequencing
de novo genome assembly (much harder with shorter reads)
Variant detection (SNPs, indels) and copy number, structural variation
Trancriptome
Whole transcriptome sequencing
Gene expression profiling
Small RNA sequencing
NGS
Next Generation Sequencing
Targeted resequencing
Exon sequencing
Epigenome
Chromatin Immunoprecipitation sequencing (Chip-Seq)
Methylation analysis
-MeDIP(Methylated DNA Immunoprecipitation )
:Anti m5C specific antibody 를 이용한 pull down
Unknown C methylation 영역 찾기에 유리

9. Next Generation Sequencer: Three Leading Platforms
454 GS FLX Titanium (Roche)
5500xl SOLiD
(AB)
HiSeq2000
(Illumina)
Bp per run
1Gb
(7Gb/7day)
300Gb Gb
( )
Read length
Av. 400 bp
35-75 bp bp
Run time
10 hr
1-7 days
4-8 days
Sample ?
2 genome
30G is 30X genome, so 1000 individual is 30T disk
With 3730s capillary, ~60 Mb per year

10. Sanger sequencing: chain termination using dideoxynucleotides
Source:

11. Pyrosequencing

12. 2 D electrophoresis/MALDI-TOF MASS: proteome 분석
2-DE by subfractionation methods
pH acidic  basic
 MW
Kidney tissue, homogenated post-mitochondrial protein, TCA/acetone, colloidal coomassie blue staining
Kim-Chulhong, PBBRC

13. Advantage of MALDI-TOF MS
빠르다. 즉 이온화 및 분석물의 검출이 모두 밀리세컨드안에 끝난다.
결과가 분명하다. 왜냐하면 근본적으로 질량 대 전하비(mass-to-charge ratio:m/z)에 근거하기 때문에 핵산의 2차 구조에 영향을 받는 전기영동이나 hybridization-array (e.g. DNA chip) 보다 정확하다.
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. Reflector (Ion Mirror)
MALDI-TOF MS
Reflector Mode : isotope peak 분리가능
Ion mirror (reflector)를 사용하여 비행거리를 증가시킴으로 resolution을 높인다
Linear Mode : resolution 이 떨어진다. R L
Flight Tube
Detector
Ion Source
4-25 kV
Reflector (Ion Mirror)
Liner mode
Kim-Chulhong, PBBRC

24. Trasfection

25. Transgenic mice

26. ㅏㅐ
Knockout mice

27. RNAi

28. Footprinting (footprint analysis)
BOX 26-1 FIGURE 1 Footprint analysis of the RNA polymerase-binding site on a DNA fragment. Separate experiments are carried out in the presence (+) and absence (–) of the polymerase.

29. Band shift assay or Electrophoretic mobility shift assay (EMSA)

30. Luciferase/reporter vector
Promoter analysis

32. Receptor binding assay

33. Radioimmunoassay (RIA)
FIGURE 23-3a Radioimmunoassay (RIA). (a) A low concentration of radiolabeled hormone (red) is incubated with 1 a fixed amount of antibody specific for that hormone or 2 a fixed amount of antibody and various concentrations of unlabeled hormone (blue). In the latter case, unlabeled hormone competes with labeled hormone for binding to the antibody; the amount of labeled hormone bound varies inversely with the concentration of unlabeled hormone present.

34. Radioimmunoassay (RIA)
FIGURE 23-3b Radioimmunoassay (RIA). (b) A radioimmunoassay for adrenocorticotropic hormone (ACTH; also called corticotropin). A standard curve of the ratio [bound]/[unbound] radiolabeled ACTH vs. [unlabeled ACTH added] (on a logarithmic scale) is constructed and used to determine the amount of (unlabeled) ACTH in an unknown sample. If an aliquot containing an unknown quantity of unlabeled hormone gives, say, a value of 0.4 for the ratio [bound]/[unbound] (see arrow), the aliquot must contain about 20 pg of ACTH.
Radioimmunoassay (RIA)