Overview of the cloning process used to isolate and amplify individual genes.

Discovery and function of restriction enzymes used in cloning. Restriction enzymes cleave DNA at certain palindromic restriction sites/sequences.

Ligases are used to rejoin pieces of DNA cut by restriction enzymes. DNA pieces with overhangs must have complementary ends to ligate together.

Vectors are circular pieces of DNA that can carry genes into the bacteria. Vectors often contain origin of replication, restriction sites, drug resistance, and bacterial promoters.

Bacteria randomly take up plasmids in the environment. Select for transformed bacteria that have taken up plasmids using antibiotic resistance such as ampicillin and kanamycin.

Find clone of interest in the library by complementation. Transform plasmids into mutant bacteria deficient in the gene and look functional rescue.

DNA sequence can be inferred from the purified and sequenced protein. Radioactive complementary DNA probes are used to hybridize to the clone of interest.

Map the gene of interest based on pedigrees. Use recombination rates to determine linkage between gene of interest and other genetic markers (small variations in the genome) within each family.

Map the gene of interest based on pedigrees. Use recombination rates to determine linkage between gene of interest and other genetic markers (small variations in the genome) within each family.

A collection of human mRNA can be reverse transcribed into a cDNA library and used in cloning. Bacteria transformed with cDNA can make human proteins.

Plasmids are purified and clones are redigested with multiple restriction enzymes and the size is determined by gel electrophoresis. Analyze clones by determining the location of the restriction sites.

Genomes can be sequenced by sequencing smaller, approximately 1000bp long, segments and combining them by overlapping regions.

PCR is a form of in vitro DNA replication used to amplify DNA segments. Primers are designed to amplify small segments of DNA. Thermostable Taq polymerase is used so it does not denature at high temperatures.

PCR has low fidelity. The process can be used to look for integrated virus segments or cancer cell. Other applications include: Single sperm typing, embryotyping, and forensics.

Brief introduction to the need to clone genes, the use of restriction enzymes to cut DNA, and PCR to amplify DNA.

General outline of the steps, components, and design of cloning.

Discussion of restriction enzymes, palindromic restriction sites, "sticky" ends, and ligation. History of Luria's experiment that discovered of restriction enzymes in bacteria. Example: EcoRI.

Bacteriophage experiment that discovered restriction enzymes. Bacteria that have restriction enzymes protect their own DNA by methylating their restriction sites.

Steps in constructing a DNA library. Cut genomic DNA into segments, insert into vectors, and transform vectors into bacteria.

Cloning a single gene by rescuing the function of the mutant using a library.

Principles and uses of gel electrophoresis. Analysis of clones using restriction enzymes and gel electrophoresis. Image of gel apparatus and UV image.

Sequencing accomplished by using dideoxynucleotide units that stop DNA polymerization in the middle of the chain. Colored dyes and gel electrophoresis are used to determine the length of the fragments and sequence of bases.

Uses of PCR in forensics and familial diseases. Components needed for PCR-template, nucleotides, primers, and heat resistant polymerase. Steps of PCR done at various temperatures-denature double helix, anneal primers, elongate chain.

Clarification of restriction enzymes, cleavage, sticky ends, and 3' OH and 5' phosphate. Linkage of the complementary sticky ends.

Circular vectors with origin of replication are used as vehicles to carry linear DNA into bacteria.

Constructing DNA library by cutting up a genome and inserting pieces into vectors. The vector containing the gene of interest can be selected by rescuing functional defects (complementation).

Issues with cloning eukaryotic genes using bacterial plasmids-different promoters and introns.

Synthesize the DNA using the protein sequence and radioactive P32. Hybridize DNA probe to recombinant library.

Cloning by linkage to genetic markers such as single nucleotide polymorphisms and recombination.

Review and clarification of DNA sequencing. Brief but good explanation.

cDNA libraries are created from mRNA to DNA, so the library does not contain introns. Fuse mammalian gene with E. coli promoter, so E. coli can make human proteins. Fuse mammalian promoters with reporter protein (can be easily assayed) such as Beta-galactosidase and GFP.

Introduction to restriction enzymes as a nucleic acid technology.

Introduction to gel electrophoresis as a nucleic acid technology.

Introduction to bacterial transformation and plasmids.

A continuation of the discussion on the method of bacterial transformation, use of plasmids, and cloning, including clone identification and cloning by complementation.

Discussion of the recent development of PCR.

Luria's experimental design and results that led to the discovery of restriction enzymes in bacteria.

Yeast as a model organism. Techniques and experiments used in the making, identifying, and characterizing yeast mutants. Test of Recessivity, Complementation Test and Epistasis Test.

Brief explanation of the use of temperature sensitive mutants to study essential genes.

Creating restriction maps using restriction enzymes. Necessary components of a bacterial expression vector that carry a gene of interest.

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Steps involved in expressing a C. elegans gene in bacteria.

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Basic cloning problem with restriction digestion and restriction mapping.

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Purification and separation by gel electrophoresis of proteins produced in bacteria. Native versus denaturing gels.

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Steps and requirements to clone a functional gene by growing mutant yeasts in various mediums.

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Basics and uses of a complementation test to characterize a group of mutants with the same phenotype.

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Examples of complementation groups for mutants and use of complementation to establish a biosynthetic pathway.

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Examples of epistasis test to determine order of enzymes and intermediates in a biochemical pathway.

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Definition and explanation of cloning, vector, library, restriction enzyme, and recombinant DNA technology.

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Example of inserting a gene into a vector using restriction enzymes and ligase. Transform vector into bacteria and select using drug resistance. Analyze clone using restriction enzymes and gel electrophoresis.

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Steps in making a genomic library of wild type yeast that can be used in cloning by complementation to rescue mutant yeast.

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Using DNA to solve crimes. Recombinant DNA technology used include: DNA sequencing, restriction digest, and gel electrophoresis.

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Constructing wild type E. coli library using restriction enzymes and plasmids. Cloning by complementation by rescue tryptophan biosynthesis deficiency.

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Analysis using gel electrophoresis. Mutations leading to changes in side chains that affect the function of the protein.

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Example of biochemical pathway with mutations in several complementation groups.

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Reagent and enzymes needed to construct a genomic library.

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Mapping restriction sites in a gene or vector using restriction enzymes and gel electrophoresis.

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Modifications used to express eukaryotic genes in bacteria.

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