1. Molecular Techniques

2. Recombinant DNA Technology
Provides ability to isolate, sequence, and manipulate individual genes derived from any type of cell
Application of the technology allows detailed studies of gene and genome structure and function

3. Restriction Endonucleases
Restriction endonucleases are enzymes that cleave DNA at specific sequences
Gel electrophoresis used after digestion to separate restriction fragments by size
Order of restriction fragments determined to make restriction map of DNA
Shows locations of cleavage sites for restriction endonucleases
REs also used for molecular cloning techniques
RFs can be isolated, cloned, and sequenced

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5. 4.14 EcoRI digestion and gel electrophoresis of l DNA
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6. 4.15 Restriction maps of l and adenovirus DNAs
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7. 4.16 Generation of a recombinant DNA molecule
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8. 4.17 Joining of DNA molecules (Part 1)
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9. 4.17 Joining of DNA molecules (Part 2)
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10. 4.18 cDNA cloning
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11. 4.19 Cloning in plasmid vectors (Part 1)
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12. 4.19 Cloning in plasmid vectors (Part 2)
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13. 4.19 Cloning in plasmid vectors (Part 3)
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14. 4.21 Expression of cloned genes in bacteria
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16. DNA Sequencing
Determination of the nucleotide sequences of genes has elucidated the structure of their protein products and the properties of DNA sequences that regulate gene expression
Performed with rapid automated systems
Requires oligonucleotide primer
Dideoxynucleoties lack the normal 3¢ hydroxyl group of deoxyribose and their incorporation stops the reaction

17. 4.20 DNA sequencing (Part 1)
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18. 4.20 DNA sequencing (Part 2)
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19. 4.20 DNA sequencing (Part 3)
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20. Nucleic Acid Hybridization
Nucleic acid hybridization is the formation of double-stranded DNA and/or RNA molecules by complementary base pairing
Southern blotting is a technique used for detection of specific genes in cellular DNA
Northern blotting, a variation of the Southern blotting technique, is used for detection of RNA instead of DNA
Recombinant DNA libraries are collections of clones that contain all the genomic or mRNA sequences of a particular cell type
DNA microarrays allow tens of thousands of genes to be analyzed simultaneously using hybridization
In situ hybridization uses radioactive or fluorescent probes to detect RNA or DNA sequences in chromosomes or intact cells

21. 4.24 Detection of DNA by nucleic acid hybridization
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22. 4.25 Southern blotting
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23. 4.26 Screening a recombinant library by hybridization
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24. 4.27 DNA microarrays (Part 1)
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25. 4.27 DNA microarrays (Part 2)
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26. 4.28 Fluorescence in situ hybridization
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27. Studying Proteins
Antibodies are proteins produced by cells of the immune system that react against molecules that the host organism recognizes as foreign substances—for example, the protein coat of a virus
Antigens are molecules against which an antibody is directed.
Monoclonal antibodies are a single species of antibody that can be produced by the culturing of clonal lines of B lymphocytes from immunized animals
Immunoblotting (also called Western blotting) is another variation of Southern blotting
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) is a method in which proteins are separated by dissolving them in a solution containing the negatively charged detergent, sodium dodecyl sulfate (SDS)
In immunoprecipitation, antibodies are used to isolate the proteins against which they are directed

28. 4.29 Western blotting
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29. 4.30 Immunoprecipitation
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30. 4.31 Immunofluorescence
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31. Studies of Eukaryote Gene Function
Understanding the function of a gene requires analysis of the gene within cells or intact organisms—not simply as a molecular clone in bacteria
Saccharomyces cerevisiae are particularly advantageous for studies of eukaryotic molecular biology
Temperature-sensitive mutants encode proteins that are functional at one temperature but not another

32. 4.32 Cloning of yeast genes
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33. Gene Transfer in Plants and Animals
Gene transfer is the introduction of foreign DNA into a cell
Transfection is a method for introducing DNA into animal cells
Liposomes are lipid vesicles that can incorporate DNA and fuse with the plasma membrane
Electroporation is the exposure of cells to a brief electric pulse that transiently opens pores in the plamsa membrane
Transient expression is a phenomenon in which DNA that has been transported to the nucleus is transcribed for several days
Animal viruses can also be used as vectors for more efficient introduction of cloned DNAs into cells
Transgenic mice carry foreign genes that have been incorporated into the germ line

34. 4.33 Introduction of DNA into animal cells
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35. 4.36 Introduction of genes into mice via embryonic stem cells (Part 2)
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36. 4.37 Introduction of genes into plant cells via the Ti plasmid
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37. Mutagenesis
In classical genetic studies, mutants are the key to identifying genes and understanding their function
Reverse genetics involves the introduction of any desired alteration into a cloned gene in order to determine the effect of the mutation on gene function
In homologous recombination, the cloned gene replaces the normal allele, so mutations introduced into the cloned gene in vitro become incorporated into the chromosomal copy of the gene.
Recombination between transferred DNA and the homologous chromosomal gene occurs frequently in yeast

38. 4.38 Mutagenesis with synthetic oligonucleotides
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39. 4.39 Gene inactivation by homologous recombination
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40. 4.40 Production of mutant mice by homologous recombination in ES cells
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41. Interfering With Gene Expression
Homologous recombination has been used to systematically inactivate, or knockout, every gene in yeast, so a collection of genome-wide yeast mutants is available for scientists to use to study the function of any desired gene
Antisense nucleic acids are RNA or single-stranded DNA complementary to the mRNA of the gene of interest
RNA interference (RNAi) is the degradation of mRNAs by short complementary double-stranded RNA molecules
It is sometimes possible to interfere directly with the function of proteins within cells through direct inhibition
Dominant inhibitory mutants are cloned DNAs encoding mutant proteins.

42. 4.41 Inhibition of gene expression by antisense RNA or DNA
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43. 4.42 RNA interference
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44. 4.43 Direct inhibition of protein function
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45. Disclaimer
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