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Overview: The DNA Toolbox

Overview: The DNA Toolbox. Sequencing of the human genome was completed by 2007 DNA sequencing has depended on advances in technology, starting with making recombinant DNA

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Overview: The DNA Toolbox

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  1. Overview: The DNA Toolbox • Sequencing of the human genome was completed by 2007 • DNA sequencing has depended on advances in technology, starting with making recombinant DNA • In recombinant DNA, nucleotide sequences from two different sources, often two species, are combined in vitro into the same DNA molecule

  2. Concept 20.1: DNA cloning yields multiple copies of a gene or other DNA segment • To work directly with specific genes, scientists prepare gene-sized pieces of DNA in identical copies, a process called DNA cloning

  3. DNA Cloning and Its Applications: A Preview • Most methods for cloning pieces of DNA in the laboratory share general features, such as the use of bacteria and their plasmids • Plasmids are small circular DNA molecules that replicate separately from the bacterial chromosome • Cloned genes are useful for making copies of a particular gene and producing a protein product

  4. Fig. 20-2 Cell containing geneof interest Bacterium 1 Gene inserted intoplasmid Bacterialchromosome Plasmid Gene ofinterest RecombinantDNA (plasmid) DNA of chromosome 2 Plasmid put intobacterial cell Recombinantbacterium 3 Host cell grown in cultureto form a clone of cellscontaining the “cloned”gene of interest Gene ofInterest Protein expressedby gene of interest Copies of gene Protein harvested Basic research andvarious applications 4 Basicresearchon protein Basicresearchon gene Gene used to alter bacteria for cleaning up toxic waste Gene for pest resistance inserted into plants Protein dissolvesblood clots in heartattack therapy Human growth hor-mone treats stuntedgrowth

  5. Fig. 20-2a Cell containing geneof interest Bacterium 1 Gene inserted intoplasmid Bacterialchromosome Plasmid Gene ofinterest RecombinantDNA (plasmid) DNA of chromosome 2 2 Plasmid put intobacterial cell Recombinantbacterium

  6. Fig. 20-2b Recombinantbacterium Host cell grown in cultureto form a clone of cellscontaining the “cloned”gene of interest 3 Protein expressedby gene of interest Gene ofInterest Copies of gene Protein harvested Basic research andvarious applications 4 Basicresearchon protein Basicresearchon gene Protein dissolvesblood clots in heartattack therapy Human growth hor-mone treats stuntedgrowth Gene for pest resistance inserted into plants Gene used to alter bacteria for cleaning up toxic waste

  7. Using Restriction Enzymes to Make Recombinant DNA • Bacterial restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites • A restriction enzyme usually makes many cuts, yielding restriction fragments • The most useful restriction enzymes cut DNA in a staggered way, producing fragments with “sticky ends” that bond with complementary sticky ends of other fragments Animation: Restriction Enzymes

  8. DNA ligase is an enzyme that seals the bonds between restriction fragments

  9. Fig. 20-3-1 Restriction site 5 3 3 5 DNA Restriction enzymecuts sugar-phosphatebackbones. 1 Sticky end

  10. Fig. 20-3-2 Restriction site 5 3 3 5 DNA Restriction enzymecuts sugar-phosphatebackbones. 1 Sticky end DNA fragment addedfrom another moleculecut by same enzyme.Base pairing occurs. 2 One possible combination

  11. Fig. 20-3-3 Restriction site 5 3 3 5 DNA Restriction enzymecuts sugar-phosphatebackbones. 1 Sticky end DNA fragment addedfrom another moleculecut by same enzyme.Base pairing occurs. 2 One possible combination DNA ligaseseals strands. 3 Recombinant DNA molecule

  12. Cloning a Eukaryotic Gene in a Bacterial Plasmid • In gene cloning, the original plasmid is called a cloning vector • A cloning vector is a DNA molecule that can carry foreign DNA into a host cell and replicate there

  13. Storing Cloned Genes in DNA Libraries • A genomic library that is made using bacteria is the collection of recombinant vector clones produced by cloning DNA fragments from an entire genome • A genomic library that is made using bacteriophages is stored as a collection of phage clones

  14. A complementary DNA (cDNA) library is made by cloning DNA made in vitro by reverse transcription of all the mRNA produced by a particular cell • A cDNA library represents only part of the genome—only the subset of genes transcribed into mRNA in the original cells

  15. Fig. 20-6-1 DNA innucleus mRNAs in cytoplasm

  16. Fig. 20-6-2 DNA innucleus mRNAs in cytoplasm Reversetranscriptase Poly-A tail mRNA Primer DNAstrand

  17. Fig. 20-6-3 DNA innucleus mRNAs in cytoplasm Reversetranscriptase Poly-A tail mRNA Primer DNAstrand DegradedmRNA

  18. Fig. 20-6-4 DNA innucleus mRNAs in cytoplasm Reversetranscriptase Poly-A tail mRNA Primer DNAstrand DegradedmRNA DNA polymerase

  19. Fig. 20-6-5 DNA innucleus mRNAs in cytoplasm Reversetranscriptase Poly-A tail mRNA Primer DNAstrand DegradedmRNA DNA polymerase cDNA

  20. Screening a Library for Clones Carrying a Gene of Interest • A clone carrying the gene of interest can be identified with a nucleic acid probe having a sequence complementary to the gene • This process is called nucleic acid hybridization

  21. A probe can be synthesized that is complementary to the gene of interest • For example, if the desired gene is – Then we would synthesize this probe … … 5 G G C T A A C T T A G C 3 C C G A T T G A A T C G 5 3

  22. The DNA probe can be used to screen a large number of clones simultaneously for the gene of interest • Once identified, the clone carrying the gene of interest can be cultured

  23. Amplifying DNA in Vitro: The Polymerase Chain Reaction (PCR) • The polymerase chain reaction, PCR, can produce many copies of a specific target segment of DNA • A three-step cycle—heating, cooling, and replication—brings about a chain reaction that produces an exponentially growing population of identical DNA molecules

  24. Fig. 20-8 3 5 TECHNIQUE Targetsequence 3 5 Genomic DNA 1 5 3 Denaturation 5 3 2 Annealing Cycle 1yields 2 molecules Primers 3 Extension Newnucleo-tides Cycle 2yields 4 molecules Cycle 3yields 8 molecules;2 molecules(in whiteboxes)match targetsequence

  25. Fig. 20-8a 5 3 TECHNIQUE Targetsequence Genomic DNA 3 5

  26. Fig. 20-8b 1 5 3 Denaturation 3 5 2 Annealing Cycle 1yields 2 molecules Primers 3 Extension Newnucleo-tides

  27. Fig. 20-8c Cycle 2yields 4 molecules

  28. Fig. 20-8d Cycle 3yields 8 molecules;2 molecules(in whiteboxes)match targetsequence

  29. Concept 20.2: DNA technology allows us to study the sequence, expression, and function of a gene • DNA cloning allows researchers to • Compare genes and alleles between individuals • Locate gene expression in a body • Determine the role of a gene in an organism • Several techniques are used to analyze the DNA of genes

  30. Gel Electrophoresis and Southern Blotting • One indirect method of rapidly analyzing and comparing genomes is gel electrophoresis • This technique uses a gel as a molecular sieve to separate nucleic acids or proteins by size • A current is applied that causes charged molecules to move through the gel • Molecules are sorted into “bands” by their size Video: Biotechnology Lab

  31. Fig. 20-9 TECHNIQUE Powersource Mixture ofDNA mol-ecules ofdifferentsizes – Cathode Anode + Gel 1 Powersource – + Longermolecules 2 Shortermolecules RESULTS

  32. Fig. 20-9a TECHNIQUE Powersource Mixture ofDNA mol-ecules ofdifferentsizes Anode Cathode – + Gel 1 Powersource – + Longermolecules 2 Shortermolecules

  33. Fig. 20-9b RESULTS

  34. In restriction fragment analysis, DNA fragments produced by restriction enzyme digestion of a DNA molecule are sorted by gel electrophoresis • Restriction fragment analysis is useful for comparing two different DNA molecules, such as two alleles for a gene • The procedure is also used to prepare pure samples of individual fragments

  35. Fig. 20-10 Normal -globin allele Normalallele Sickle-cellallele 175 bp Large fragment 201 bp DdeI DdeI DdeI DdeI Largefragment Sickle-cell mutant -globin allele 376 bp 201 bp175 bp Large fragment 376 bp DdeI DdeI DdeI (a) DdeI restriction sites in normal and sickle-cell alleles of -globin gene (b) Electrophoresis of restriction fragments from normal and sickle-cell alleles

  36. A technique called Southern blotting combines gel electrophoresis of DNA fragments with nucleic acid hybridization • Specific DNA fragments can be identified by Southern blotting, using labeled probes that hybridize to the DNA immobilized on a “blot” of gel

  37. Fig. 20-11 TECHNIQUE Heavyweight Restrictionfragments I II III Nitrocellulosemembrane (blot) DNA + restriction enzyme Gel Sponge I Normal-globinallele II Sickle-cellallele III Heterozygote Papertowels Alkalinesolution 2 1 3 Preparation of restriction fragments DNA transfer (blotting) Gel electrophoresis Radioactively labeledprobe for -globin gene Probe base-pairswith fragments I II III I II III Fragment fromsickle-cell-globin allele Film overblot Fragment fromnormal -globin allele Nitrocellulose blot 4 5 Probe detection Hybridization with radioactive probe

  38. Fig. 20-11a TECHNIQUE Heavyweight Restrictionfragments I II III DNA + restriction enzyme Nitrocellulosemembrane (blot) Gel Sponge I Normal-globinallele II Sickle-cellallele III Heterozygote Papertowels Alkalinesolution 2 3 1 Preparation of restriction fragments Gel electrophoresis DNA transfer (blotting)

  39. Fig. 20-11b Radioactively labeledprobe for -globin gene Probe base-pairswith fragments I II III I II III Fragment fromsickle-cell-globin allele Film overblot Fragment fromnormal -globin allele Nitrocellulose blot 5 4 Hybridization with radioactive probe Probe detection

  40. DNA Sequencing • Relatively short DNA fragments can be sequenced by the dideoxy chain termination method • Modified nucleotides called dideoxyribonucleotides (ddNTP) attach to synthesized DNA strands of different lengths • Each type of ddNTP is tagged with a distinct fluorescent label that identifies the nucleotide at the end of each DNA fragment • The DNA sequence can be read from the resulting spectrogram

  41. Fig. 20-12 TECHNIQUE Primer Deoxyribonucleotides Dideoxyribonucleotides(fluorescently tagged) DNA(template strand) dATP ddATP dCTP ddCTP dTTP ddTTP DNA polymerase dGTP ddGTP DNA (template strand) Labeled strands Shortest Longest Directionof movementof strands Longest labeled strand Detector Laser Shortest labeled strand RESULTS Last baseof longestlabeledstrand Last baseof shortestlabeledstrand

  42. Fig. 20-12a TECHNIQUE DNA(template strand) Primer Deoxyribonucleotides Dideoxyribonucleotides(fluorescently tagged) dATP ddATP dCTP ddCTP dTTP ddTTP DNA polymerase dGTP ddGTP

  43. Fig. 20-12b TECHNIQUE DNA (template strand) Labeled strands Shortest Longest Directionof movementof strands Longest labeled strand Detector Laser Shortest labeled strand RESULTS Last baseof longestlabeledstrand Last baseof shortestlabeledstrand

  44. Analyzing Gene Expression • Nucleic acid probes can hybridize with mRNAs transcribed from a gene • Probes can be used to identify where or when a gene is transcribed in an organism

  45. Studying the Expression of Single Genes • Changes in the expression of a gene during embryonic development can be tested using • Northern blotting • Reverse transcriptase-polymerase chain reaction • Both methods are used to compare mRNA from different developmental stages

  46. Northern blotting combines gel electrophoresis of mRNA followed by hybridization with a probe on a membrane • Identification of mRNA at a particular developmental stage suggests protein function at that stage

  47. Reverse transcriptase-polymerase chain reaction (RT-PCR) is quicker and more sensitive • Reverse transcriptase is added to mRNA to make cDNA, which serves as a template for PCR amplification of the gene of interest • The products are run on a gel and the mRNA of interest identified

  48. Fig. 20-13 TECHNIQUE 1 cDNA synthesis mRNAs cDNAs Primers 2 PCR amplification -globingene 3 Gel electrophoresis Embryonic stages RESULTS 1 2 3 4 5 6

  49. In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism

  50. Fig. 20-14 50 µm

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