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DNA Technology and Genomics
Recombinant DNA
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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
Bacterial genetics
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Nucleoid:
– region in bacterium
densely packed with
DNA (no membrane)
Plasmids:
– small circles of DNA
Reproduction by binary
fission (asexual)
Bacterial DNA-transfer
processes
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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
Bacterial Plasmids
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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
Recombination of E. coli
Bacterial plasmids in gene cloning
Clone genes for
insertion into
organisms
 Clone proteins for
medical/
pharmaceutical
purposes
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DNA Cloning
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Restriction enzymes (endonucleases)
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Restriction site
–
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short extensions of restriction fragments
DNA ligase
–
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segments of DNA cut by restriction enzymes in a
reproducable way
Sticky end
–
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recognition sequence for a particular restriction
enzyme
Restriction fragments
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in nature, these enzymes protect bacteria from
intruding DNA
they cut up the DNA (restriction)
very specific
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)
Eukaryotic Gene Cloning
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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
Genomic Libraries
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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
DNA Analysis
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PCR (polymerase chain
reaction)
Gel electrophoresis
Restriction fragment
analysis (RFLPs)
Southern blotting
DNA sequencing
Practical DNA Technology Uses
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Diagnosis of disease
Human gene therapy
Pharmaceutical products
– Vaccines
– Hormones
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Forensics
– Crime scene analysis of DNA
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Animal husbandry
(transgenic organisms)
– “Pharm” animals
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Genetic engineering in plants
– Disease/ pest resistance
Polymerase chain reaction (PCR)
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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.
DNA Analysis
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Gel electrophoresis:
– separates nucleic
acids or proteins on
the basis of size or
electrical charge
creating DNA bands of
the same length
Restriction fragment analysis
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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
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Southern blotting: process
that reveals sequences and
the RFLPs in a DNA sequence
DNA Fingerprinting
Southern Blotting
DNA Sequencing
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Determination of nucleotide
sequences
– Dideoxy Chain-Termination
Method (Sanger Method)
– Whole-genome approach
(Venter and Celera
Genomics)
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Genomics: the study of
genomes based on DNA
sequences
Human Genome Project
– Begun in 1990; largely
completed by 2003
Genomics
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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
Analyzing Gene Expression
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Northern Blotting
– Gel electrophoresis done
with labeling probes to
determine function
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RT-PCR
– Uses reverse
transcriptase and PCR
– Compares gene
expression between
different samples
Studying Gene Interaction
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DNA Microassay
– Many DNA
fragments on a glass
slide or chip
– Can be tested for
interaction with other
genes marked with
fluorescent markers
Determining Gene Function
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In vitro mugagenasis
– Disable certain genes and
observe consequences
– Mutations “knock out” certain
genes
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RNA interference (RNAi)
– RNA used to block translation
of certain genes
Transposons
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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”
Eukaryotic 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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Transposable Elements
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Transposons
– Move w/in a genome by
DNA intermediate
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Barbara McClintock
(1940’s and 50’s)
– “Jumping genes”
– Researched the location of
colored kernels in maize
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Retrotransposons
– Move by means of a RNA
intermediate
Multigene Families
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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
– Similar- α-globin and β-globin
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Pseudogenes- nonfunctional nucleotide sequences (very
similar to functional genes)
Genome Evolution
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Duplications of
chromosomes
– Unequal crossing over
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Duplication and
divergence of DNA
segments
– Ancestral globin gene
 present day α-globin
and β-globin genes
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Rearranging genes
– Exon duplication/ exon
shuffling
– Transposable elements