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Studying and Manipulating Genomes

Studying and Manipulating Genomes. Chapter 16 Hsueh-Fen Juan Oct 30, 2012. 16.1 Cloning DNA. Researchers cut up DNA from different sources, then paste the resulting fragments together Cloning vectors can carry foreign DNA into host cells. Cut and Paste. Restriction enzymes

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Studying and Manipulating Genomes

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  1. Studying and Manipulating Genomes Chapter 16 Hsueh-Fen Juan Oct 30, 2012

  2. 16.1 Cloning DNA • Researchers cut up DNA from different sources, then paste the resulting fragments together • Cloning vectors can carry foreign DNA into host cells

  3. Cut and Paste • Restriction enzymes • Bacterial enzymes that cut DNA wherever a specific nucleotide sequence occurs • 之所以叫限制酶，是因為它的發現來自於，科學家發現有的細菌不怕噬菌體，因為噬菌體DNA一注入，這種細菌的酶會馬上去分解此噬菌體DNA的特定序列部位，「限制」噬菌體的感染，故名限制酶。 • Single-stranded DNA tails produced by the same restriction enzyme base-pair together • DNA ligase bonds “sticky ends” together • Recombinant DNA • Composed of DNA from two or more organisms

  4. Animation: Base-pairing of DNA fragments

  5. Animation: Restriction enzymes

  6. restriction enzyme (cut) DNA ligase (paste) mix C Matching sticky ends of different fragments base-pair with each other, regardless of the source of the DNA. A A restriction enzyme recognizes a specific base sequence in DNA (red boxes, 6個非4個). For this and many other enzymes, the sequence is the same in the 5 to 3 direction on both strands. B Researchers use restriction enzymes to cut DNA from different sources into fragments. Fragments with identical sticky ends are mixed together. DDNA ligase joins the fragments of DNA where they overlap. Molecules of recombinant DNA are the result. Making Recombinant DNA Stepped Art Fig. 16-2, p. 242

  7. DNA Cloning • DNA cut into fragments by restriction enzymes is inserted into cloning vectors (plasmids) cut with the same enzyme • Cloning vectors with foreign DNA are placed in host cells, which divide and produce many clones, each with a copy of the foreign DNA

  8. Cloning Vectors

  9. DNA Cloning F The result? Recombinant plasmids that carry foreign DNA. These plasmids are introduced into host cells, which divide to form clones. enzyme E The DNA fragments and the cut plasmid are mixed. The sticky ends of different fragments that base- pair are bonded by DNA ligase. A A restriction enzyme cuts a specific base sequence everywhere it occurs in DNA. C The DNA fragments have sticky ends. B The same enzyme cuts the same sequence in plasmid DNA. D The plasmid DNA also has sticky ends. Fig. 16-4, p. 243

  10. Animation: Formation of recombinant DNA

  11. cDNA Cloning • Complementary DNA (cDNA) • DNA made from an mRNA template • 在真核即「只包含外顯子部分的DNA」 • Reverse transcriptase transcribes mRNA to DNA, forming a hybrid molecule • DNA polymerase builds a double-stranded DNA molecule that can be cloned

  12. cDNA Cloning

  13. 16.2 From Haystacks to Needles • DNA libraries and the polymerase chain reaction (PCR) help researchers isolate particular DNA fragments

  14. DNA Libraries • Genome • The entire set of genetic material of an organism • DNA libraries are sets of cells containing various cloned DNA fragments • Genomic libraries (all DNA in a genome) • cDNA libraries (all active genes in a cell)

  15. Animation: How to make cDNA

  16. Probes • Probe • A fragment of DNA labeled with a tracer • Used to find a specific clone carrying DNA of interest in a library of many clones • Nucleic acid hybridization • Base pairing between DNA from different sources • A probe hybridizes with the targeted gene

  17. Nucleic Acid Hybridization using a Radioactive Probe

  18. A Individual bacterial cells from a DNA library are spread over the surface of a solid growth medium. The cells divide repeatedly and form colonies—clusters of millions of genetically identical daughter cells. B A piece of special paper pressed onto the surface of the growth medium will bind some cells from each colony. C The paper is soaked in a solution that ruptures the cells and releases their DNA. The DNA clings to the paper in spots mirroring the distribution of colonies. D A probe is added to the liquid bathing the paper. The probe hybridizes with (sticks to) only the spots of DNA that contain complementary base sequences. E The bound probe makes a spot. Here, one radioactive spot darkens x-ray film. The position of the spot on the film is compared to the positions of all the original bacterial colonies. Cells from the colony that made the spot are cultured, and the DNA they contain is harvested. Fig. 16-5, p. 244

  19. Animation: Use of a radioactive probe

  20. Big-Time Amplification: PCR • Polymerase chain reaction (PCR) • A cycled reaction that uses a heat-tolerant form of DNA polymerase (Taq polymerase) to produce billions of copies of a DNA fragment • 因為PCR過程要重複加溫降溫，而一般的DNA聚合酶在高溫(能解開螺旋的溫度)時通常會被破壞，而一種耐高溫細菌(Thermus aquaticus, aka Taq)的DNA聚合酶就很適合用在這裡

  21. PCR • DNA to be copied is mixed with DNA polymerase, nucleotides and primers that base-pair with certain DNA sequences • Cycles of high and low temperatures break and reform hydrogen bonds between DNA strands, doubling the amount of DNA in each cycle

  22. A DNA template (purple) is mixed with primers (red), free nucleotides, and heat-tolerant Taq DNA polymerase. PCR B When the mixture is heated, DNA strands separate. When it is cooled, some primers hydrogen-bond to the template DNA. CTaq polymerase uses the primers to initiate synthesis, and complementary strands of DNA form. The first round of PCR is now complete. D The mixture is heated again, and all of the DNA separates into single strands. When the mixture is cooled, some of the primers hydrogenbond to the DNA. E Taq polymerase uses the primers to initiate DNA synthesis, and complementary strands of DNA form. The second round of PCR is complete. Each round can double the number of DNA molecules. After 30 rounds, the mixture contains huge numbers of DNA fragments, all copies of the template DNA. Fig. 16-6, p. 245

  23. Animation: Polymerase chain reaction (PCR)

  24. 16.3 DNA Sequencing • DNA sequencing reveals the order of nucleotide bases in a fragment of DNA

  25. DNA Sequencing • DNA is synthesized with normal nucleotides and dideoxynucleotides (雙去氧核醣核苷酸) tagged with different colors • When a tagged base is added, DNA synthesis stops; fragments of all lengths are made • 雙去氧核醣核苷酸的3’沒有氧，因此無法讓下一個核苷酸的5’接上來，因此會停在那裏 • Electrophoresis (電泳) separates the fragments of DNA, each ending with a tagged base, by length • Order of colored bases is the sequence of DNA

  26. Tagged Dideoxynucleotides • Each base is labeled with a different color

  27. A The fragment of DNA to be sequenced is mixed with a primer, DNA poly- merase, and nucleotides. The mixture also includes the four dideoxynucleotides labeled with four different colored pigments. DNA Sequencing B The polymerase uses the DNA as a tem- plate to synthesize new strands again and again. Synthesis of each new strand stops when a dide- oxynucleotideis added. D An electro- phoresis gel sepa- rates the fragments into bands according to length. All fragments in each band end with the same dideoxynucleotide; thus, each band is the color of that dideoxynucleotide. C At the end of the reaction, there are many truncated copies of the DNA template in the mixture. E A computer detects and records the color of each band on the gel. The order of colors of the bands represents the sequence of the template DNA. Fig. 16-8, p. 246

  28. Animation: Automated DNA sequencing

  29. Next Generation Sequencing (NGS) Trends in Genetics 2008, 24 (3): 142–149.

  30. 16.4 DNA Fingerprinting • One individual can be distinguished from all others on the basis of DNA fingerprints

  31. DNA Fingerprints • DNA fingerprint • A unique array of DNA sequences used to identify individuals • Short tandem repeats (STRs) 短縱列重複序列 • 人類約有99%基因相同，剩下1%差異很大 • Many copies of the same 2- to 10-base-pair sequences in a series along a chromosome 是很短且重複排在一起的片段 • Types and numbers of STRs vary greatly among individuals

  32. Creating DNA Fingerprints • PCR is used to amplify DNA from regions of several chromosomes that have STRs • Electrophoresis is used to separate the fragments and create a unique DNA fingerprint • DNA fingerprints have many applications • Legal cases, forensics, population studies

  33. DNA Fingerprints: A Forensics Case

  34. Animation: DNA fingerprinting

  35. 16.5 Studying Genomes • Comparing the sequence of our genome with that of other species is giving us insights into how the human body works

  36. The Human Genome Project • Automated DNA sequencing and PCR allowed human genome projects to sequence the 3 billion bases in the human genome • 28,976 genes have been identified, but not all of their products or functions are known

  37. Sequencing the Human Genome

  38. Genomics • Genomics: The study of genomes • Structural genomics 專注於基因所扁馬出來之蛋白質的三度空間立體結構 • Comparative genomics 專注於比較不同物種間的基因差異 • Analysis of the human genome yields new information about genes and how they work • Applications in medicine and other fields • Example:APOA5 mutations and triglycerides

  39. DNA Chips • DNA chips • Microarrays of many different DNA samples arranged on a glass plate • Used to compare patterns of gene expression among cells of different types or under different conditions • May be used to screen for genetic abnormalities, pathogens, or cancer

  40. DNA Chips and Gene Expression

  41. Principle of cDNA microarrays

  42. Principle of cDNA microarrays

  43. Principle of oligonucleotide arrays

  44. Construction of Oligonucleotide Arrays

  45. Construction of Oligonucleotide Arrays

  46. Microarray Images Produced with a Pin-and-loop Arrayer

  47. 16.6 Genetic Engineering • Genetic engineering • A laboratory process by which deliberate changes are introduced into an individual’s genome • The most common genetically modified organisms are bacteria and yeast • Used in research, medicine, and industry • Example: To produce human insulin

  48. GMOs • Genetically modified organisms (GMOs) • Individuals containing modified genes from the same species or a different species • Transgenic organisms • Individuals containing genes transferred from a different species (also GMOs) • Example: Bacteria with jellyfish genes

  49. Transgenic Organisms • Bacteria with jellyfish genes

  50. 16.7 Designer Plants • Genetically engineered crop plants are widespread in the United States

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