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

DNA Technologies and Genomics(DNA 기술과 유전체학 )

Chapter 18

단백질체학에서의 단백질 미세배열 .

그림 18.1. 살인사건 해결의 실마리가 되었던 Snowball 이름의 고양이 .

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

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

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

그림 18.2. DNA 절편을 세균 플라스미드에 클로닝하는 방법 .

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

그림 18.3. 제한효소 EcoRI 을 이용한 재조합 DNA 의 형성 .

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

그림 18.4. 플라스미드 벡터에 유전자 클로닝 .

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

그림 18.5. 특정 DNA 서열을 식별하기 위한 DNA 혼성화 .

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

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

그림 18.6. 중합효소연쇄반응

(PCR).

그림 18.7. 아가로오스 겔 전기영동에 의한 DNA 절편 분리 .

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

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

그림 18.8. β- 글로빈 유전자의 제한효소 절단위치의 차이 .

그림 18.9. 서던 블랏 분석법 .

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

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

그림 18.10. PCR 를 이용한 STR 유전자 좌에 대한 DNA

지문분석 .

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 )

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

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 .

배아를 대리모에 착상시킨다 .

배아가 성장하여 자손이 태어난다 .

자손 생쥐를 교배한다 .

Genetically Engineered Mouse

그림 18.12. 집쥐의 성장호르몬 유전자를 주입하여 유전자 조작된 거대 생쥐 ( 우측 ).

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 )

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

Cloned Sheep

“Dolly”

그림 18.13. 복제양 돌리 .

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

Crown Gall Tumor

그림 18.14. 캘리포니아 후추나무의 근두암종 .

그림 18.15. 뿌리혹세균의 Ti 플라스미드를 이용한 식물 형질전환 .

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

GMO Tobacco

그림 18.16. 반딧불이의 발광효소 유전자를 도입한 담배 .

GMO Rice

그림 18.17. 베타 - 카로틴을 함유하도록 유전자 변형된 쌀 .

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

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

Genome Analysis

Genomics • Analyzes organization of complete genome and

gene networks

Human Genome Project took 13 years (2003)• Revolutionizing biology and evolutionary

understanding

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

그림 18.18. DNA 염기서열 결정을 위한 dideoxy (Sanger)

방법 .

Genomic Analyses (1)

Structural genomics • Sequence genomes to locate genes and funtional

sequenes

Functional genomics • Studies functions of genes and other parts of

genome

Genomic Analyses (2)

Whole-genome shotgun method • Breaks genome into many DNA fragments

• Computers assemble genome based on overlapping sequences

그림 18.19. 전체 유전체 샷건 염기서열 결정법 .

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

Human Genome

3.2 billion (32 억 ) 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

Genome Analysis

Data mining• Gene functions

• Genome organization

• Expression controls

Comparative genomics (with other organisms)• Tests evolutionary hypotheses

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

그림 18.20. DNA microarray 분석 .

Proteomics

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

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