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Definition: DNA in which genes from 2 different sources are linked
Genetic engineering: direct manipulation of genes for practical purposes
Biotechnology: manipulation of organisms or their components to perform practical tasks or provide useful products

 Nucleoid:
– region in bacterium densely packed with
DNA (no membrane)
 Plasmids:
– small circles of DNA
 Reproduction by binary fission (asexual)

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Transformation
– genotype alteration by the uptake of naked, foreign DNA from the environment (Griffith expt.)
Transduction
– phages that carry bacterial genes from
1 host cell to another
– generalized ~ random transfer of host cell chromosome
– specialized ~ incorporation of prophage
DNA into host chromosome
Conjugation
– direct transfer of genetic material; cytoplasmic bridges
– pili; sexual

 Small, circular, self-replicating DNA separate from the bacterial chromosome
 F (fertility) Plasmid: codes for the production of sex pili (F+ or F-)
 R (resistance) Plasmid: codes for antibiotic drug resistance

 Clone genes for insertion into organisms
 Clone proteins for medical/ pharmaceutical purposes

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Restriction enzymes (endonucleases)
– in nature, these enzymes protect bacteria from intruding DNA
– they cut up the DNA (restriction)
– very specific
Restriction site
– recognition sequence for a particular restriction enzyme
Restriction fragments
– segments of DNA cut by restriction enzymes in a reproducable way
Sticky end
– short extensions of restriction fragments
DNA ligase
– enzyme that can join the sticky ends of DNA fragments
Cloning vector
– DNA molecule that can carry foreign DNA into a cell and replicate there (usually bacterial plasmids)

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Isolation of cloning vector
(bacterial plasmid) & gene-source
DNA (gene of interest)
Insertion of gene-source DNA into the cloning vector using the same restriction enzyme; bind fragmented DNA w/ DNA ligase
Introduction of cloning vector into cells (transformation by bacterial cells)
Cloning of cells (and foreign genes)
Identification of cell clones carrying the gene of interest

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Cloned genes from a genome are stored in a “genomic library”
Recombinant fragments in bacteria or phages
Complimentary DNA
(cDNA) Library
– mRNA extracted
– Reverse transcriptase makes a complimentary strand of gene

 PCR (polymerase chain reaction)
 Gel electrophoresis
 Restriction fragment analysis (RFLPs)
 Southern blotting
 DNA sequencing

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Diagnosis of disease
Human gene therapy
Pharmaceutical products
– Vaccines
– Hormones
Forensics
– Crime scene analysis of DNA
Animal husbandry
(transgenic organisms)
– “Pharm” animals
Genetic engineering in plants
– Disease/ pest resistance

 Amplification of any piece of DNA without cells (in vitro)
 Materials: heat, DNA polymerase, nucleotides, single-stranded DNA primers
 Applications: fossils, forensics, prenatal diagnosis, etc.

 Gel electrophoresis:
– separates nucleic acids or proteins on the basis of size or electrical charge creating DNA bands of the same length

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Restriction fragment length polymorphisms (RFLPs)
– Differences in restriction fragment patterns on homologous chromosomes
– Occur in noncoding DNA sequences
– Serve as inheritable genetic markers
Southern blotting: process that reveals sequences and the RFLPs in a DNA sequence
DNA Fingerprinting

 Determination of nucleotide sequences
– Dideoxy Chain-Termination
Method (Sanger Method)
– Whole-genome approach
(Venter and Celera
Genomics)
 Genomics: the study of genomes based on DNA sequences
 Human Genome Project
– Begun in 1990; largely completed by 2003

 The National Center for
Biotechnology
Information (NCBI)
– Created a database of gene sequences created by the
Human Genome Project and other sequencing endeavors
– Genbank
– BLAST software allows for comparison of sequences

 Northern Blotting
– Gel electrophoresis done with labeling probes to determine function
 RT-PCR
– Uses reverse transcriptase and PCR
– Compares gene expression between different samples

 DNA Microassay
– Many DNA fragments on a glass slide or chip
– Can be tested for interaction with other genes marked with fluorescent markers

 In vitro mugagenasis
– Disable certain genes and observe consequences
– M utations “knock out” certain genes
 RNA interference (RNAi)
– RNA used to block translation of certain genes

 transposable genetic element; piece of
DNA that can move from location to another in a cell’s genome
– chromosome to plasmid, plasmid to plasmid, etc.)
– “jumping genes”

 98.5% of all DNA does not code for proteins, rRNA, or tRNA
 Most is repetitive DNA
 44% is made of transposable elements

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Transposons
– Move w/in a genome by
DNA intermediate
Barbara McClintock
(1940’s and 50’s)
– “Jumping genes”
– Researched the location of colored kernels in maize
Retrotransposons
– Move by means of a RNA intermediate

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In the human genome, ½ of coding DNA is in multigene families
– Collections of identical or very similar genes
– Identical- ribosomal RNA molecules
– Similarα-globin and β-globin
Pseudogenes- nonfunctional nucleotide sequences (very similar to functional genes)

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Duplications of chromosomes
– Unequal crossing over
Duplication and divergence of DNA segments
– Ancestral globin gene
 present day α-globin and β-globin genes
Rearranging genes
– Exon duplication/ exon shuffling
– Transposable elements