Molecular Techniques

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

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

4.14 EcoRI digestion and gel electrophoresis of l DNA

4.15 Restriction maps of l and adenovirus DNAs

4.16 Generation of a recombinant DNA molecule

4.17 Joining of DNA molecules (Part 1)

4.17 Joining of DNA molecules (Part 2)

4.18 cDNA cloning

4.19 Cloning in plasmid vectors (Part 1)

4.19 Cloning in plasmid vectors (Part 2)

4.19 Cloning in plasmid vectors (Part 3)

4.21 Expression of cloned genes in bacteria

4.20 DNA sequencing (Part 1)

4.20 DNA sequencing (Part 2)

4.20 DNA sequencing (Part 3)

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

4.24 Detection of DNA by nucleic acid hybridization

4.25 Southern blotting

4.26 Screening a recombinant library by hybridization

4.27 DNA microarrays (Part 1)

4.27 DNA microarrays (Part 2)

4.28 Fluorescence in situ hybridization

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

4.29 Western blotting

4.30 Immunoprecipitation

4.31 Immunofluorescence

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

4.32 Cloning of yeast genes

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

4.33 Introduction of DNA into animal cells

4.36 Introduction of genes into mice via embryonic stem cells (Part 2)

4.37 Introduction of genes into plant cells via the Ti plasmid

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

4.38 Mutagenesis with synthetic oligonucleotides

4.39 Gene inactivation by homologous recombination

4.40 Production of mutant mice by homologous recombination in ES cells

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.

4.41 Inhibition of gene expression by antisense RNA or DNA

4.42 RNA interference

4.43 Direct inhibition of protein function

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