Chapter 15 Biotechnology (Sections 15.1 - 15.5)

1. Chapter 15Biotechnology (Sections 15.1 - 15.5)

2. 15.1 Personal DNA Testing • Personal DNA testing companies identify a person’s unique array of single-nucleotide polymorphisms (SNPs) – some of which are related to risk of diseases such as Alzheimer’s • Personal genetic testing may soon revolutionize medicine by allowing physicians to customize treatments on the basis of an individual’s genetic makeup

3. Personal DNA Testing • This chip reveals which versions of 906,600 SNPs occur in the individual’s DNA

4. ABC Video: DNA Mystery: Human Chimeras

5. ABC Video: Family Ties: Paternity Testing

6. 15.2 Cloning DNA • Researchers use restriction enzymes to cut up DNA, then bond the fragments together using DNA ligase • Fragments with complementary tails (“sticky ends”) stick together when their matching tails base-pair • restriction enzyme • Bacterial enzyme used to cut specific nucleotide sequences in DNA

7. Recombinant DNA • DNA fragments from different organisms combine to make a hybrid molecule: recombinant DNA • recombinant DNA • A DNA molecule that contains genetic material from more than one organism

8. Making Recombinant DNA (1) • A restriction enzyme recognizes a specific base sequence in DNA from any source

9. Making Recombinant DNA (2) • The enzyme cuts DNA from two sources into fragments • The enzyme leaves sticky ends

10. Making Recombinant DNA (3) • When DNA fragments from the two sources are mixed together, matching sticky ends base-pair with each other

11. Making Recombinant DNA (4) • DNA ligase joins the base-paired DNA fragments • Molecules of recombinant DNA result

12. Making Recombinant DNA DNA ligase (paste) mix restriction enzyme (cut) A restriction enzyme recognizes a specific base sequence (orange boxes) in DNA from any source. When the DNA fragments from the two sources are mixed together, matching sticky ends base-pair with each other. DNA ligase joins the base-paired DNA fragments. Molecules of recombinant DNA are the result. 1 3 4 The enzyme cuts DNA from two sources into fragments. This enzyme leaves sticky ends. 2 Fig. 15.2, p. 220

13. DNA ligase (paste) mix restriction enzyme (cut) A restriction enzyme recognizes a specific base sequence (orange boxes) in DNA from any source. When the DNA fragments from the two sources are mixed together, matching sticky ends base-pair with each other. DNA ligase joins the base-paired DNA fragments. Molecules of recombinant DNA are the result. 1 3 4 The enzyme cuts DNA from two sources into fragments. This enzyme leaves sticky ends. 2 Making Recombinant DNA Stepped Art Fig. 15.2, p. 220

14. Animation: Restriction Enzymes

15. DNA Cloning • DNA cloning mass produces specific DNA fragments • Fragments to be copied are inserted into plasmids or other cloning vectors and inserted into host cells such as bacteria • Host cells divide and make identical copies (clones) of the foreign DNA

16. Key Terms • DNA cloning • Set of procedures that uses living cells to make many identical copies of a DNA fragment • plasmid • Of many bacteria and archaeans, a small ring of nonchromosomal DNA replicated independently of the chromosome • cloning vector • A DNA molecule that can accept foreign DNA, be transferred to a host cell, and get replicated in it

17. Plasmid Cloning Vectors

18. Plasmid Cloning Vectors Kpn l Sph l Pst l Bam Hl Eco RI Sal l Acc l Xho l Xba l Bst XI Sac l Not l pDrive Cloning Vector 3.85 kb A B Fig. 15.3, p. 220

19. Plasmid Cloning Vectors Fig. 15.3a, p. 220

20. Plasmid Cloning Vectors Fig. 15.3b, p. 220

21. DNA Cloning

22. DNA Cloning A A restriction enzyme cuts a specific base sequence in chromosomal DNA and in a plasmid cloning vector. chromosomal DNA fragments chromosomal DNA B A fragment of chromosomal DNA and the plasmid base-pair at their sticky ends. DNA ligase joins the two pieces of DNA. recombinant plasmid plasmid cloning vector cut plasmid C The recombinant plasmid is inserted into a host cell. When the cell multiplies, it makes multiple copies of the plasmids. Fig. 15.4, p. 221

23. A A restriction enzyme cuts a specific base sequence in chromosomal DNA and in a plasmid cloning vector. chromosomal DNA fragments chromosomal DNA B A fragment of chromosomal DNA and the plasmid base-pair at their sticky ends. DNA ligase joins the two pieces of DNA. recombinant plasmid plasmid cloning vector cut plasmid C The recombinant plasmid is inserted into a host cell. When the cell multiplies, it makes multiple copies of the plasmids. DNA Cloning Stepped Art Fig. 15.4, p. 221

24. ANIMATION: Formation of recombinant DNA To play movie you must be in Slide Show Mode PC Users: Please wait for content to load, then click to play Mac Users: CLICK HERE

25. cDNA Cloning • Researchers who study eukaryotic genes and their expression work with mRNA transcripts of genes • RNA can’t be cloned directly – reverse transcriptase,a viral enzyme, is used to transcribe single-stranded RNA into complementary DNA (cDNA)for cloning

26. Key Terms • reverse transcriptase • A viral enzyme that uses mRNA as a template to make a strand of cDNA • cDNA • DNA synthesized from an RNA template by the enzyme reverse transcriptase

27. cDNA Cloning (1) • A strand of cDNA, is assembled on an mRNA template:

28. cDNA Cloning (2) • DNA polymerase removes RNA and copies the cDNA into a second strand of DNA, resulting in a double-stranded DNA copy of the original mRNA:

29. Key Concepts • DNA Cloning • Researchers routinely make recombinant DNA by cutting and pasting together DNA from different species • Plasmids and other vectors can carry foreign DNA into host cells

30. ANIMATION: Base-pairing of DNA fragments To play movie you must be in Slide Show Mode PC Users: Please wait for content to load, then click to play Mac Users: CLICK HERE

31. ANIMATION: How to Make cDNA To play movie you must be in Slide Show Mode PC Users: Please wait for content to load, then click to play Mac Users: CLICK HERE

32. 15.3 From Haystacks to Needles • DNA libraries are sets of cells that host cloned DNA fragments • A genomic library collectively contains all DNA in a genome • A cDNA library contains only those genes being expressed at the time the mRNA was harvested • DNA libraries and the polymerase chain reaction (PCR) help researchers isolate particular DNA fragments

33. Key Terms • DNA library • Collection of cells that host different fragments of foreign DNA, often representing an organism’s entire genome • genome • An organism’s complete set of genetic material

34. Isolating Genes • Researchers use probesthat match a targeted DNA sequence to identify cells with a specific DNA fragment • A probe mixed with DNA from a library base-pairs with (hybridizes to) the targeted gene • Base pairing between nucleic acids from different sources is called nucleic acid hybridization

35. Key Terms • probe • Short fragment of DNA labeled with a tracer such as a radioactive isotope • Designed to hybridize with a nucleotide sequence of interest • nucleic acid hybridization • Base-pairing between DNA or RNA from different sources

36. Nucleic Acid Hybridization

37. Nucleic Acid Hybridization 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 descendant 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 (base-pairs) with 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 is compared to the positions of the original bacterial colonies. Cells from the colony that made the spot are cultured, and the DNA they contain is harvested. Fig. 15.5, p. 222

38. ANIMATION: Use of a radioactive probe To play movie you must be in Slide Show Mode PC Users: Please wait for content to load, then click to play Mac Users: CLICK HERE

39. PCR • The polymerase chain reaction (PCR) uses primers and heat-resistant DNA polymerase to mass-produce a particular section of DNA without having to clone it in living cells • polymerase chain reaction (PCR) • Method that rapidly generates many copies of a specific section of DNA • primer • Short, single strand of DNA designed to hybridize with a DNA fragment

40. Multiplication by PCR • PCR can be used on any sample of DNA with at least one molecule of a target sequence • Essentially any sample containing DNA can be used, even one sperm, a hair left at a crime scene, or a mummy • Each cycle of a PCR reaction doubles the number of copies of a section of DNA – thirty cycles can make a billion copies

41. Two Rounds of PCR (1) • DNA template is mixed with primers, nucleotides, and heat-tolerant Taq DNA polymerase

42. Two Rounds of PCR (1) targeted section DNA template (blue) is mixed with primers (pink), nucleotides, and heat-tolerant Taq DNA polymerase. 1 Fig. 15.6.1, p. 223

43. Two Rounds of PCR (2) • When the mixture is heated, the double-stranded DNA template separates into single strands – when it is cooled, primers base-pair with template DNA

44. Two Rounds of PCR (2) When the mixture is heated, the double-stranded DNA template separates into single strands. When it is cooled, some of the primers base-pair with the template DNA. 2 Fig. 15.6.2, p. 223

45. Two Rounds of PCR (3) • Taq polymerase begins DNA synthesis at primers, so complementary DNA strands form on single-stranded templates

46. Two Rounds of PCR (3) Taq polymerase begins DNA synthesis at the primers, so complementary strands of DNA form on the single-stranded templates. 3 Fig. 15.6.3, p. 223

47. Two Rounds of PCR (4) • The mixture is heated again; double-stranded DNA separates into single strands • When it is cooled, primers basepair with old and new DNA strands

48. Two Rounds of PCR (4) The mixture is heated again, and the double-stranded DNA separates into single strands. When it is cooled, some of the primers base-pair with the template DNA. The copied DNA also serves as a template. 4 Fig. 15.6.4, p. 223

49. Two Rounds of PCR (5) • Each round of PCR reactions doubles the number of copies of the targeted DNA section

50. Two Rounds of PCR (5) Each round of PCR reactions can double the number of copies of the targeted DNA section. 5 Fig. 15.6.5, p. 223