1. DNA Technologies and Genomics (DNA 기술과 유전체학)
Chapter 18

2. 단백질체학에서의 단백질 미세배열.

3. 그림 18.1. 살인사건 해결의 실마리가 되었던 Snowball 이름의 고양이.

4. Biotechnology Biotechnology
Any technique applied to biological systems to manipulate processes
DNA technologies isolate, purify, analyze and manipulate DNA sequences
DNA fingerprinting used in forensics
Genetic engineering uses DNA technologies to alter genes for practical purposes

5. 18.1 DNA Cloning
Bacterial enzymes (restriction endonucleases) form the basis of DNA cloning
Bacterial plasmids illustrate the use of restriction enzymes in cloning
DNA libraries contain collections of cloned DNA fragments
Polymerase chain reaction (PCR) amplifies DNA in vitro

6. Recombinant DNA DNA cloning provides many copies of a gene
Used for research or manipulation
Recombinant DNA contains DNA from multiple sources joined together
Recombinant plasmids containing gene of interest can be cloned in E. coli

7. 그림 18.2. DNA 절편을 세균 플라스미드에 클로닝하는 방법.

8. Endonucleases
Restriction enzymes (endunucleases) cut DNA at specific sequences in restriction sites
Restriction fragments (제한효소절편) result
Sticky ends (점착성 말단) have unpaired bases at cuts which will hydrogen bond
Ligase stitches together paired sticky ends

9. 그림 18.3. 제한효소 EcoRI을 이용한 재조합 DNA의 형성.

10. Plasmid Cloning Vectors
Engineered to contain gene of interest and sorting genes
Sorting genes identify E. coli with cloned plasmid
E. coli with appropriate plasmid are ampicillin resistant and blue-white screened on X-gal

11. 그림 플라스미드 벡터에 유전자 클로닝.

12. DNA Hybridization (혼성화)
Uses nucleic acid probe to identify gene of interest in set of clones
Probe has tag for detection
Identified colony produces large quantities of cloned gene

13. 그림 18.5. 특정 DNA 서열을 식별하기 위한 DNA 혼성화.

14. DNA Libraries Genomic libary (유전체 라이브러리)
Clones containing every sequence in a genome
Used to isolate genes or DNA sequences
Complementary DNA (cDNA) library
DNA sequences made from expressed RNA
mRNA extracted from cell
Reverse transcriptase makes cDNA
Removes introns for genetic engineering

15. Polymerase Chain Reaction
Polymerase chain reaction (PCR) (중합효소연쇄반응)
Produces many sequence copies without host cloning
Amplifies known DNA sequences for analysis
Only copies sequence of interest
Primers bracket sequence
Agarose gel electrophoresis
Separates fragments by size and charge
Gel molecular sieve

16. 그림 중합효소연쇄반응(PCR).

17. 그림 18.7. 아가로오스 겔 전기영동에 의한 DNA 절편 분리.

18. 18.2 Application of DNA Technologies
DNA technologies are used in molecular testing for many human genetic diseases
DNA fingerprinting used to identify human individuals and individuals of other species
Genetic engineering uses DNA technologies to alter the genes of a cell or organism
DNA technologies and genetic engineering are a subject of public concern

19. RFLPs Restriction fragment length polymorphisms (제한효소절편길이다형성)
DNA sequence length changes due to varying restriction sites from same region of genome
Sickle cell anemia has RFLPs
Southern blot analysis uses electrophoresis, blot transfer, and labeled probes to identify RFLPs
Alternative is PCR and electrophoresis

20. 그림 18.8. β-글로빈 유전자의 제한효소 절단위치의 차이.

21. 그림 서던 블랏 분석법.

22. DNA Fingerprinting (지문감식)
Distinguishes between individuals
Uses PCR at multiple loci within genome
Each locus heterozygous or homzygous for short tandem repeats (STR)
PCR amplifies DNA from STR
Number of gel electrophoresis bands shows amplified STR alleles
13 loci commonly used in human DNA fingerprinting

23. Forensics and Ancestry (법의학과 가계분석)
Forensics compares DNA fingerprint from sample to suspect or victim
Usually reported as probability DNA came from random individual
Common alleles between children and parents used in paternity tests
Same principle used to determine evolutionary relationships between species

24. 그림 18.10. PCR를 이용한 STR 유전자 좌에 대한 DNA 지문분석.

25. Genetic Engineering Transgenic organisms
Modified to contain genes from external source
Expression vector has promoter in plasmid for production of transgenic proteins in E. coli
Example: Insulin
Protocols to reduce risk of escape (노출 위험성을 최소화하는 안전성 지침)

26. Animal Genetic Engineering
Transgenic animals used in research, correcting genetic disorders, and protein production
Germ-line cell (생식세포) transgenes can be passed to offspring (somatic can not)
Embryonic germ-line cells cultured in quantity, made into sperm or eggs
Stem cells

27. 그림 18.11. 배아 생식세포를 이용하여 생쥐의 배아에 유전자 도입
배아에서 채취한 생식세포에 주사 또는 전기천공법으로 원하는 유전자를 주입한다.
유전자가 주입된 세포를 배양해 순수 형질전환 세포집단을 얻는다
형질전환 세포를 초기배아(포배)에 주입한다.
그림 배아 생식세포를 이용하여 생쥐의 배아에 유전자 도입
배아를 대리모에 착상시킨다.
배아가 성장하여 자손이 태어난다.
자손 생쥐를 교배한다.

28. Genetically Engineered Mouse
그림 집쥐의 성장호르몬 유전자를 주입하여 유전자 조작된 거대 생쥐(우측).

29. Gene Therapy Attempts to correct genetic disorders
Germ-line gene therapy (생식세포 유전자치료) can’t be used on humans
Somatic gene therapy (체세포 유전자치료) used in humans
Mixed results in humans
Successes for ADA (탈아미노효소 결핍증) and sickle-cell
Deaths from immune response and leukemia-like conditions (백혈병과 비슷한 증세)

30. Animal Genetic Engineering
“Pharm” animals produce proteins for humans
Usually produced in milk for harmless extraction
Cloned mammals produced by implantation of diploid cell fused with denucleated egg cell
Low cloning success rate
Increased health defects in clones
Gene expression regulation abnormal

31. Cloned Sheep
“Dolly”
그림 복제양 돌리.

32. Plant Genetic Engineering
Has been highly successful
Increased resistance to environmental effects and pathogens
Plant “pharms” and increased nutrition
Callus formation
Ti (tumor inducing) plasmid from crown gall disease (근두암종병) used as vector
Transforming DNA (T DNA) genes expressed

33. Crown Gall Tumor
그림 캘리포니아 후추나무의 근두암종.

34. 그림 18.15. 뿌리혹세균의 Ti 플라스미드를 이용한 식물 형질전환.

35. GMO Concerns
Genetically modifed organisms (GMOs) are transgenic and raise certain concerns
Effect on environment
Interbreeding with or harming natural species
Cartagena Protocol on Biosafety (생물안전성에 관한 카르타지나 협정) provides rules on GMOs
Stringent laboratory standards for transgenic organisms
No bacterial “escapes” from labs

36. 그림 18.16. 반딧불이의 발광효소 유전자를 도입한 담배.
GMO Tobacco
그림 반딧불이의 발광효소 유전자를 도입한 담배.

37. 그림 18.17. 베타-카로틴을 함유하도록 유전자 변형된 쌀.
GMO Rice
그림 베타-카로틴을 함유하도록 유전자 변형된 쌀.

38. 18.3 Genome Analysis
DNA sequencing techniques are based on DNA replication
Structural genomics determines the complete DNA sequence of genomes
Functional genomics focuses on the functions of genes and other parts of the genome

39. 18.3 (cont.)
Studying the array of expressed proteins is the next level of genomic analysis
Systems biology is the study of the interactions between all the components of an organism

40. Genome Analysis Genomics Human Genome Project took 13 years (2003)
Analyzes organization of complete genome and gene networks
Human Genome Project took 13 years (2003)
Revolutionizing biology and evolutionary understanding

41. DNA Sequencing Used for small DNA sequences to genomes
Dideoxy (Sanger) method of sequencing
Dideoxyribonucleotides have –H bound to 3’ C instead of –OH
DNA polymerases place dideoxyribonucleotides in DNA, stops replication
Polyacrylamide gel separates strands varying by one nucleotide

42. 그림 18.18. DNA 염기서열 결정을 위한 dideoxy (Sanger) 방법.

43. Genomic Analyses (1) Structural genomics Functional genomics
Sequence genomes to locate genes and funtional sequenes
Functional genomics
Studies functions of genes and other parts of genome

44. Genomic Analyses (2) Whole-genome shotgun method
Breaks genome into many DNA fragments
Computers assemble genome based on overlapping sequences

45. 그림 전체 유전체 샷건 염기서열 결정법.

46. Functional Genomics Bioinformatics
Analysis of large data sets
Uses biology, computer science, mathematics
Identify open reading frames with start and stop codons, sophisticated algorithms for introns
Sequence similarity searches
Genomics revealed many unknown genes
Many genes similar between evolutionarily distant organisms

47. Human Genome 3.2 billion (32억) base pairs
Between 20,000 and 25,000 genes
About 100,000 proteins
Due to alternative splicing and protein processing
Protein coding only 2% of genome
24% introns
50% repeat sequences of no known function

48. Genome Analysis Data mining
Gene functions
Genome organization
Expression controls
Comparative genomics (with other organisms)
Tests evolutionary hypotheses

49. DNA Microarrays DNA microarrays (chips)
About 20 nucleotide-long DNA probe sequences
cDNA probes made from isolated mRNA
Probes red or green from different cell states
cDNA from each cell state hybridize with complementary sequences on chip
Used to determine how expression changes in normal and cancer cells
Also used to detect mutations

50. 그림 DNA microarray 분석.

51. Proteomics Proteome Proteomics (study of proteome)
Complete set of proteins expressed by genome
Larger than genome in eukaryotes
Proteomics (study of proteome)
Protein microarrays (chips) similar to DNA microarrays
Use antibodies to bind to proteins

52. Systems Biology Studies organisms as a whole
Investigates networks of genes, proteins, and biochemistry
Combines genomics and proteomics with response to environment
Complex data analysis and computer models limiting factors