1. Chapter 14 The Techniques of Molecular Genetics
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2. Chapter Outline
Basic Techniques used to Identify, Amplify, and Clone Genes
Construction and Screening of DNA Libraries
The Molecular Analysis of DNA, RNA, and Protein
The Molecular Analysis of Genes and Chromosomes
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3. Basic Techniques Used to Identify, Amplify, and Clone Genes
Recombinant DNA, gene cloning, and DNA amplification techniques allow scientists to isolate and characterize essentially any DNA sequence from any organism.
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4. Gene Cloning
Gene cloning is the isolation and amplification of a given gene.
A recombinant DNA molecule is a DNA molecule made by joining two or more different DNA molecules.
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5. Amplification of a Gene In Vivo
A minichromosome carrying the gene of interest is produced in the test tube.
The recombinant minichromosome is introduced into a host cell (such as E. coli), and the host cell replicates the minochromosome.
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6. Amplification of a Gene In Vitro
Short DNA strands complementary to DNA sequences on either side of the gene of interest are synthesized.
These short DNA strands are used to initiate the amplification of the gene by a heat-stable DNA polymerase in the polymerase chain reaction (PCR).
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7. Restriction Endonucleases
Restriction endonucleases make site-specific cuts in DNA.
The nucleotide sequences are called restriction sites.
Restriction endonucleases protect bacteria from foreign DNA.
Bacteria protect endogenous restriction sites by methylation.
Restriction enzymes commonly recognize palindromic sequences.
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10. Structure of an EcoRI-DNA Complex
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11. Many Restriction Endonucleases Make Staggered Cuts
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12. When DNA is cleaved with a restriction endonuclease that makes staggered cuts, all of the resulting restriction fragments have complementary single-stranded termini.
The complementary single-stranded termini can hydrogen bond with each other and be joined together by DNA ligase.
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13. Construction of Recombinant DNA Molecules In Vitro
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17. Plasmid Vectors
Circular, double-stranded circular DNA molecules present in bacteria.
Range from 1 kb to over 200 kb.
Replicate autonomously.
Many carry antibiotic-resistance genes, which can be used as selectable markers.
Many useful cloning vectors were derived from plasmid pBR322.
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18. Bacteriophage Vectors
Most bacteriophage cloning vectors have been constructed from the phage  chromosome.
The central one-third (about 15 kb) of the  chromosome contains genes required for lysogeny but not for lytic growth.
This portion of the chromosome can be excised and replaced with foreign DNA.
The foreign DNA inserted must be kb.
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19. Cosmid Vectors Hybrids between plasmids and the phage  chromosome.
Replicate autonomously in E. coli.
Can be packaged in vitro into phage  heads.
Accept inserts of kb.
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20. Phagemid Vectors
Contain components from phage chromosomes and plasmids.
Replicate in E. coli as double-stranded plasmids.
Addition of a helper phage causes the phagemid to switch to the phage mode of replication, resulting in the packaging of single-stranded DNA into phage heads.
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22. The Blue-White Color Test
The E. coli lacZ gene encodes -galactosidase.
-galactosidase converts the colorless substrate Xgal into a blue product.
Cells with -galactosidase activity produce blue colonies when grown on Xgal; cells lacking -galactosidase activity produce white colonies.
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24. Eukaryotic and Shuttle Vectors
Because different organisms use different origins of replication and regulatory signals, different cloning vectors must be used in different species.
Special cloning vectors can replicate in other prokaryotes and in eukaryotes.
Shuttle vectors can replicate in E. coli and in another species.
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25. Yeast Artificial Chromosomes (YACs)
Genetically engineered yeast minichromosomes.
Accept foreign DNA inserts of kb.
Contain a yeast origin of replication, yeast centromere, two yeast telomeres, a selectable marker, and a polycloning site.
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26. BACs and PACs
Bacterial artificial chromosomes (BACs) have been constructed from bacterial fertility (F) factors.
Bacteriophage P1 artificial chromosomes (PACs) have been constructed from bacteriophage P1 chromosomes.
BACs and PACs accept kb inserts and are less complex than YACs.
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27. The Polymerase Chain Reaction (PCR)
Synthetic nucleotides complementary to known flanking sequences are used to prime enzymatic amplification of the sequence of interest.
Three repeated steps
Denaturation of genomic DNA (92-95°C)
Annealing of denatured DNA to oligonucleotide primers (50-60°C)
Replication of the DNA segment between the sites complementary to the primers (70-72°C)
Amplification occurs exponentially; each cycle doubles the number of molecules of the sequence of interest.
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28. Taq Polymerase
DNA polymerase from Thermus aquaticus is used for PCR because it is heat-stable.
Taq polymerase lacks proofreading activity, so errors are introduced into the amplified DNA at low but significant frequencies.
When high fidelity is required, heat-stable polymerases with proofreading activity are used (Pfu or Tli).
Taq is amplifies fragments of DNA larger than a few thousand base pairs inefficiently.
For amplification of long segments of DNA (up to 35 kb), Tfl polymerase is used.
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29. Applications of PCR
Diagnosis of inherited human diseases (e.g., prenatal diagnosis).
Identification of individuals in forensic cases from small DNA samples.
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33. Key Points
The discovery of restriction endonucleases—enzymes that recognize and cleave DNA in a sequence-specific manner—allowed scientists to produce recombinant DNA molecules in vitro.
DNA sequences can be inserted into small, self-replicating DNA molecules called cloning vectors and amplified by replication in vivo after being introduced into living cells by transformation.
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34. Key Points
The polymerase chain reaction (PCR) can be used to amplify specific DNA sequences in vitro.
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35. Construction and Screening of DNA Libraries
DNA libraries can be constructed and screened for genes and other sequences of interest.
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36. DNA Libraries
A genomic DNA library is a set of DNA clones collectively containing the entire genome of an organism.
A cDNA library contains the coding regions of the expressed genes of an organism. It is made of complementary DNA (cDNA) synthesized from RNA by reverse transcriptase.
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37. Cloning Restriction Fragments with Complementary Single-Stranded Ends
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39. Amplification of Recombinant DNA
Antibiotic-sensitive recipient cells are transformed with the recombinant DNA molecule.
Transformed cells are selected by growth under conditions requiring the presence of a selectable marker present on the recombinant DNA molecule (usually an antibiotic).
The recombinant DNA molecule is amplified by the host cell.
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40. Synthesis of Double-Stranded cDNAs from mRNA
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41. Screening DNA Libraries for Genes of Interest
Genetic Selection—searching for a DNA sequence that restores the wild-type phenotype to a mutant organism.
Molecular hybridization is based on the hybridization of similar DNA sequences.
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42. Colony Hybridization
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43. Key Points
DNA libraries can be constructed that contain complete sets of genomic DNA sequences or DNA copies (cDNAs) of mRNAs in an organism.
Specific genes or other DNA sequences can be isolated from DNA libraries by genetic complementation or by hybridization to labeled nucleic acid probes containing sequences of known function.
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44. The Molecular Analysis of DNA, RNA, and Protein
DNA, RNA, or protein molecules can be separated by gel electrophoresis, transferred to membranes, and analyzed by various procedures.
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45. Analysis of DNAs by Southern Blot Hybridization
DNA molecules can be separated by size by gel electrophoresis using agarose or acrylamide gels for larger and small DNA molecules, respectively.
DNA molecules can then be transferred from the gel onto a nitrocellulose or nylon membrane using a technique called a Southern blot.
DNA on the membrane can be hybridized with DNA probes.
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46. Agarose Gel Electrophoresis
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51. Southern Blot: Transferring DNA from the Gel to a Membrane
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52. Identification of a Specific Fragment by Southern Blot Hybridization
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53. Detection of Wild-Type and Mutant Alleles of the Cystic Fibrosis Gene
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54. Analysis of RNAs by Northern Blot Hybridizations
The Northern Blot procedure is nearly identical to Southern blotting, except
RNA is sensitive to degradation by RNases; contamination with these enzymes must be prevented.
RNA molecules contain extensive secondary structure and must be kept denatured during electrophoresis.
Northern blots are useful in studies of gene expression.
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55. Northern Blot Hybridization Data (RNA from roots, leaves, and flowers of A. thaliana)
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56. Analysis of RNAs by Reverse Transcriptase-PCR (RT-PCR)
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57. Analysis of Proteins by Western Blot Techniques
Polypeptides are separated by polyacrylamide gel electrophoresis in the presence of a detergent that denatures the proteins.
Proteins are transferred from the gel to a nitrocellulose membrane.
Individual proteins are detected with antibodies.
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58. Key Points
DNA restriction fragments and other small DNA molecules can be separated by agarose or acrylamide gel electrophoresis and transferred to nylon membranes to produce DNA gel blots called Southern blots.
The DNAs on Southern blots can be hybridized to labeled DNA probes to detect sequences of interest by autoradiography.
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59. Key Points
When RNA molecules are separated by gel electrophoresis and transferred to membranes for analysis, the resulting RNA gel blots are called northern blots.
RNA molecules can be detected and analyzed by reverse transcriptase-PCR (RT-PCR).
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60. Key Points
When proteins are transferred from gels to membranes and detected with antibodies, the products are called western blots.
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61. The Molecular Analysis of Genes and Chromosomes
The sites at which restriction enzymes cleave DNA molecules can be used to construct physical maps of the molecules; however, nucleotide sequences provide the ultimate physical maps of DNA molecules.
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62. Mapping Restriction Enzyme Cleavage Sites
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63. Restriction maps reflect true physical distances (unlike genetic maps).
Restriction maps can be combined with other molecular techniques to construct physical maps of entire genomes.
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64. Techniques Necessary for Sequencing DNA
Restriction enzymes to prepare homogenous samples of specific segments of chromosomes.
Gel electrophoresis procedures able to resolve DNA fragments differing in length by a single nucleotide.
Gene-cloning techniques allowing preparation of large quantities of a DNA molecule.
Sanger sequencing Technique is used to determine nucleotide sequences.
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65. DNA Sequencing A population of DNA fragments is generated.
One end is common to all fragments (the 5’ end of the sequencing primer).
The other end terminates at all possible positions (the 3- terminus).
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66. 2',3'-Dideoxyribonucleoside Triphosphates
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67. Automated DNA Sequencing
Fluorescent dyes are used for detection of DNA chains instead of radioactive isotopes.
Products of all four chain terminator reactions are separated through a single gel or capillary tube.
Photocells detect fluorescence of the dyes as they pass through the gel or capillary tube.
Output of the photocell is directly transferred to a computer for analysis.
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68. Automated Sanger DNA Sequencing
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73. Key Points
Detailed physical maps of DNA molecules can be prepared by identifying the sites that are cleaved by various restriction endonucleases.
The nucleotide sequences of DNA molecules provide the ultimate physical maps of genes and chromosomes.
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