DNA Technologies and Genomics

1. DNA Technologies and Genomics Chapter 18

2. Why It Matters • Snowball: Key to a Murder

3. 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

4. 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

5. 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

6. Cloning DNA Fragments

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

8. Restriction Enzyme EcoRI

9. 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

10. Plasmid Cloning

11. DNA Hybridization • Uses nucleic acidprobe to identify gene of interest in set of clones • Probe has tag for detection • Identified colony produces large quantities of cloned gene

12. DNA Hybridization

13. DNA Libraries • Genomic libary • 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

14. Polymerase Chain Reaction • Polymerase chain reaction (PCR) • 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

15. Polymerase Chain Reaction

16. Agarose Gel Electrophoresis

17. 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

18. RFLPs • Restriction fragment length polymorphisms • 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

19. Sickle-Cell RFLPs

20. Southern Blot Analysis

21. DNA Fingerprinting • 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

22. Forensics and Ancestry • 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

23. DNA Fingerprint

24. 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

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

26. Transgenic Mice

27. Genetically Engineered Mouse

28. Gene Therapy • Attempts to correct genetic disorders • Germ-line gene therapy can’t be used on humans • Somatic gene therapy used in humans • Mixed results in humans • Successes for ADA and sickle-cell • Deaths from immune response and leukemia-like conditions

29. 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

30. Cloned Sheep • “Dolly”

31. 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 used as vector • Transforming DNA (T DNA) genes expressed

32. Crown Gall Tumor

33. Ti Plasmid and Transgenic Plants

34. Rhizobium radiobacter disarmed so cannot induce tumors Plant cell (not to scale) Nucleus T DNA with gene of interest integrated into plant cell chromosome Regenerated transgenic plant Fig. 18-15b, p. 389

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 provides rules on GMOs • Stringent laboratory standards for transgenic organisms • No bacterial “escapes” from labs

36. GMO Tobacco

37. GMO Rice

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 • 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. Dideoxy (Sanger) Method

43. Genomic Analyses (1) • Structural 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. Whole-Genome Shotgun Sequencing

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 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. DNA Microarray Analysis