19.1 Techniques of Molecular Genetics Have Revolutionized Biology Recombinant DNA Technology—Genetic Engineering—Biotechnology: Locating, isolating, altering, and studying DNA segments Biotechnology: Using recombinant DNA technology to develop new biological products

19.2 Molecular Techniques Are Used to Isolate, Recombine, and Amplify Genes

Cutting and Joining DNA Fragments Restriction enzymes: recognizing and cutting DNA at specific nucleotide sequences Palindromic sequences Immune system of bacteria Type II restriction enzyme: most useful enzyme By adding methyl groups to the recognition sequence to protect itself from being digested by its own enzyme in bacteria

Cutting and Joining DNA Fragments Cohesive ends: fragments with short, single-stranded overhanging ends Blunt ends: even-length ends from both single strands

19.2a Restriction enzymes make double-stranded cuts in DNA, producing cohesive, or sticky, ends.

19.2b Restriction enzymes make double-stranded cuts in DNA, producing cohesive, or sticky, ends.

Viewing DNA Fragments Gel electrophoresis-separation of DNA fragments by size through a gel medium Smaller fragments migrate faster

19.3 Gel electrophoresis can be used to separate DNA molecules on the basis of their size and electrical charge. [Photograph courtesy of Carol Eng.]

19.3d Gel electrophoresis can be used to separate DNA molecules on the basis of their size and electrical charge. [Photograph courtesy of Carol Eng.]

Viewing DNA Fragments Locating DNA fragments with Southern blotting and probes Probe: DNA or RNA with a base sequence complementary to a sequence in the gene of interest Is usually labeled for easy detection Radioactive P32 Fluorescent tag

19.4 Southern blotting and hybridization with probes can locate a few specific fragments in a large pool of DNA.

Cloning Genes Gene cloning: amplifying a specific piece of DNA via a bacteria cell Cloning vector: a replicating DNA molecule attached with a foreign DNA fragment to be introduced into a cell Has features that make it easier to insert DNA and select for presence of vector in cell. Origin of replication Antibiotic resistance gene Cloning site

19.5 An idealized cloning vector has an origin of replication, one or more selectable markers, and one or more unique restriction sites.

Cloning Genes Plasmid vectors Linkers: synthetic DNA fragments containing restriction sites Transformation of host cells with plasmids Selectable markers are used to confirm whether the cells have been transformed or not.

19. 6 The pUC19 plasmid is a typical cloning vector 19.6 The pUC19 plasmid is a typical cloning vector. It contains a cluster of unique restriction sites, an origin of replication, and two selectable markers— an ampicillin-resistance gene and a lacZ gene.

19.7 A foreign DNA fragment can be inserted into a plasmid with the use of restriction enzymes.

19.8 The lacZ gene can be used to screen bacteria containing recombinant plasmids. A special plasmid carries a copy of the lacZ gene and an ampicillin- resistance gene. [Photograph: Cytographics/Visuals Unlimited.]

19.8 (part 2) The lacZ gene can be used to screen bacteria containing recombinant plasmids. A special plasmid carries a copy of the lacZ gene and an ampicillin-resistance gene. [Photograph: Cytographics/Visuals Unlimited.]

Amplifying DNA fragments with the polymerase chain reaction (PCR) Taq polymerase: stable DNA polymerase at high temperature Researcher designs specific oligonucleotide primers that serve as the ends of the amplified fragment Very similar to replication

19.11 The polymerase chain reaction is used to amplify even very small samples of DNA.

19.12 The Bt toxin gene,which encodes an insecticide,was isolated from bacteria and transferred to tobacco plants.

19.3 Molecular Techniques Can Be Used to Find Genes of Interest

Gene Libraries Gene library: a collection of clones containing all the DNA fragments from one source Creating a genomic DNA library cDNA library: consisting only of those DNA sequences that are transcribed into mRNA Creating a cDNA library

19.15a A cDNA library contains only those DNA sequences that are transcribed into mRNA.

19.15b A cDNA library contains only those DNA sequences that are transcribed into mRNA.

Gene Libraries Screening DNA libraries Plating clones of the library Probing plated colonies or plaques

19.16 Genomic and cDNA libraries can be screened with a probe to find the gene of interest.

In Situ Hybridization DNA probes used to determine the chromosomal location and to visualize a gene while it is in a cell May also be used to detect localization of mRNA expression in multicellular organisms.

19.17a With in situ hybridization, DNA probes are used to determine the cellular or chromosomal location of a gene or its product. (a) A probe with green fluorescence is specific to chromosome 7, revealing a deletion on one copy of chromosome 7. [Addenbrookes Hospital/Photo Researchers.]

19.17b With in situ hybridization, DNA probes are used to determine the cellular or chromosomal location of a gene or its product. (b) In situ hybridization is used to detect the presence of mRNA from the tailless gene in a Drosophila embryo. [Courtesy of L. Tsuda.]

Positional Cloning Isolating genes on the basis of their position on a genetic map

19.21 The gene for cystic fibrosis was located by positional cloning.

19.4 DNA Sequences Can Be Determined and Analyzed Restriction Fragment Length Polymorphisms (RFLPs) Some DNA fragments have different restriction sites due to mutation for the same restriction enzyme, which causes polymorphisms within a population.

19.23 Restriction fragment length polymorphisms are genetic markers that can be used in mapping.

DNA Sequencing Sanger’s dideoxy-sequencing method Dideoxyribonucleoside triphosphate (ddNTP) lacks a 3′-oh group, which terminates DNA synthesis.

19.26 The dideoxy method of DNA sequencing is based on the termination of DNA synthesis.

DNA Fingerprinting (DNA Profiling) Microsatellites: variable number of copies of repeat sequences possessed by many organisms, which can be amplified by PCR Combined with RFLP analysis to form more thorough fingerprint

19.5 Molecular Techniques Are Increasingly Used to Analyze Gene Function Forward genetics: Begins with a phenotype to a gene that encodes the phenotype Reverse genetics: Begins with a gene of unknown function, first inducing mutations and then checking the effect of the mutation on the phenotype

19.5 Molecular Techniques Are Increasingly Used to Analyze Gene Function Site-directed mutagenesis Reverse genetics: creating mutation in particular DNA sequences, and then studying the effects of these mutation on the organisms

19.5 Molecular Techniques Are Increasingly Used to Analyze Gene Function Silencing genes with RNAi Using RNAi for the treatment of human disease: lowering ApoB with RNAi Model Genetic Organism The mouse, Mus musculus

19. 36 The mouse Mus musculus is a model genetic organism 19.36 The mouse Mus musculus is a model genetic organism. [Chromosome photograph courtesy of Ellen C. Akeson and Muriel T. Davisson, The Jackson Laboratory, Bar Harbor, Maine.]

19.6 Biotechnology Harnesses the Power of Molecular Genetics Pharmaceuticals Human insulin Specialized bacteria Agricultural products Oligo nucleotide drugs Genetic testing Gene therapy Direct transfer of genes into humans to treat disease