DNA Technology and Genomics
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Transcript of DNA Technology and Genomics
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Recombinant DNA
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
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Bacterial genetics Nucleoid:
– region in bacterium densely packed with DNA (no membrane)
Plasmids: – small circles of DNA
Reproduction by binary fission (asexual)
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Bacterial DNA-transfer processes
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
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Bacterial Plasmids 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
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Recombination of E. coli
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Bacterial plasmids in gene cloning
Clone genes for insertion into organisms
Clone proteins for medical/ pharmaceutical purposes
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DNA Cloning 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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Eukaryotic Gene Cloning 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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Genomic Libraries
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
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DNA Analysis PCR (polymerase chain
reaction) Gel electrophoresis Restriction fragment
analysis (RFLPs) Southern blotting DNA sequencing
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Practical DNA Technology Uses 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
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Polymerase chain reaction (PCR)
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.
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DNA Analysis
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 analysis 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
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Southern Blotting
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DNA Sequencing 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
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Genomics 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
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Analyzing Gene Expression Northern Blotting
– Gel electrophoresis done with labeling probes to determine function
RT-PCR– Uses reverse
transcriptase and PCR– Compares gene
expression between different samples
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Studying Gene Interaction DNA Microassay
– Many DNA fragments on a glass slide or chip
– Can be tested for interaction with other genes marked with fluorescent markers
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Determining Gene Function
In vitro mugagenasis– Disable certain genes and
observe consequences– Mutations “knock out” certain
genes RNA interference (RNAi)
– RNA used to block translation of certain genes
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Transposons 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”
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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
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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Multigene Families 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
Pseudogenes- nonfunctional nucleotide sequences (very similar to functional genes)
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Genome Evolution
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