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Transcript of DNA Technology Problem: A chromosome can be millions of base pairs long. How do you isolate and...
![Page 1: DNA Technology Problem: A chromosome can be millions of base pairs long. How do you isolate and study a single gene? Scientists had to find out how to.](https://reader035.fdocuments.us/reader035/viewer/2022062714/56649d365503460f94a0e161/html5/thumbnails/1.jpg)
DNA TechnologyProblem:A chromosome can be millions of base pairs long.How do you isolate and study a single gene?Scientists had to find out how to cut, paste, and copy DNA.
Fragile X chromosome
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Cutting and Pasting DNA• 1962 – DNA cutting enzymes isolated from
E. coli = restriction endonucleases, aka “molecular scissors”
- left “sticky ends”
• Arthur Kornberg identified the pasting
mechanism for DNA- an enzyme called ligase- made artificial viral DNA loops
Werner Arber
Arthur KornbergMade “life in a
test tube”
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Restriction Enzymes
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Gene CloningCurrent applications:A gene that originated in one organism can be used to giveanother organism a new metabolic capability.
Useful protein products can beharvested in large quantities from bacterial cultures.
Can also be used for basic research on genes or proteins.
Molecular cloning – isolating a defined sequence of DNA and making multiple copies of it “in vivo”.
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Using Reverse Transcriptase to Synthesize
cDNA• Reverse transcriptase – an RNA-dependent DNA polymerase
• Encoded by retroviruses – copy viral RNA into DNA prior to integration into host
•Transcribes both ss RNA and ss DNA (needs a primer)•Degrades the RNA from RNA-DNA hybrids.
•Used to copy RNA into complementary DNAs (cDNAs).
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Making cDNA for a Eukaryotic Gene Using Reverse
Transcriptase
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Cloning a Eukaryotic Gene in a Bacterial Plasmid
X-gal is hydrolyzed by B-galactosidaseto yield a blue product.If a plasmid has foreign DNA insertedinto its lac Z gene, then the colony ofcells producing it will be white.
Can synthesize a nucleic acid probe -cDNA (complementary to the gene of interest) labeled with a radioactive isotope or a fluorescent tag.
A “shotgun” approach
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Using a Nucleic Acid Probe to Identify a Cloned Gene
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Cloning a eukaryotic gene into a prokaryotic cell: problems and
solutions• Differences in gene expression (ex: promoters and other
DNA control sequences).- Solution: expression vector - contains a prokaryotic promoter just before the site where the eukaryotic gene is inserted.
• Presence of long introns in most eukaryotic genes (bacterial cells do not have RNA-splicing machinery).
- Solution: make artificial eukaryotic genes that lack introns. -extract processed RNA from the eukaryotic nucleus (no
introns)-use reverse transcriptase to make cDNA transcripts of this
RNA.-cDNA is attached to vector DNA for replication inside a cell.-vector provides a bacterial promoter and any other
necessary control elements
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Using Eukaryotic Cells for Cloning: Yeast
• Avoids eukaryotic-prokaryotic incompatibility• Yeast – single-celled, easy to grow, have plasmids• Scientists have constructed vectors called
yeast artificial chromosomes (YACs) -have an origin for DNA replication, a centromere, and two telomeres-behave normally in mitosis – foreign DNA cloned as the yeast cell divides-can carry more DNA than a plasmid vector
• Problem: many eukaryotic proteins have to be modified before they are functional (addition of carbohydrate or lipid groups)
-Yeast cells may not be able to modify the protein correctly
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Artificial Chromosomes
Mammalian satellite DNA-basedartificial chromosomes (SATACs)
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Use of host cells from animal or plant cell cultures
• Many kinds of eukaryotic cells can take up foreign DNA – but not very efficiently
• Scientists have developed a variety of more aggressive methods:
-electroporation – apply a brief electrical pulse to a solution
containing cells. Creates a temporary hole in the CM thru which DNA can enter.-inject DNA directly into cells with a microscopic needle-in plants, can attach DNA to microscopic particles of metals and fire the particles into cells with a “gene gun”
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DNA Libraries• DNA fragments cloned at random into a plasmid
vector - the majority of genetic information will be included in the mixture of bacteria (“shotgun” approach)
• Cultures of the bacteria, collectively contain all the genes and are called a library.
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cDNA Libraries
• Partial genomic library• Produced using the mRNA molecules
isolated from a cell.• Contains only the genes that are
expressed (transcribed) within the cell.
• Advantage – can study the genes responsible for specialized functions in specific cell types.
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The Polymerase Chain Reaction (PCR)
• A technique of quickly amplifying DNA without using cells
• DNA contains the sequence “targeted” for copying
• A heat-resistant DNA polymerase is added (isolated from bacteria living in hot springs!)
• Plus a supply of all four nucleotides and primers
• Primers are short, synthetic molecules of single-stranded DNA complementary to the ends of the targeted DNA
• Each cycle takes only about 5 minutes to complete
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DNA Analysis and Genomics• Genomics – sequencing and studying whole sets of genes and
their interactions - to make comparisons between cells, individuals, and species.
• Gel electrophoresis – separates nucleic acids and proteins based on size and charge
• Pure samples of these bands can be recovered from the gel and retain their biological activity.
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Restriction Fragment Analysis by Southern
Blotting1. Restriction enzyme applied.2. Gel separation.3. Blot onto nitrocellulose paper.4. Ss-DNA probes added.5. Hybridization.6. Bands identified ARG.
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RFLP
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Restriction Fragment Length Polymorphisms
(RFLPs)• Used to find differences in noncoding
sequences of DNA.
• Can serve as genetic markers for a particular location (locus) in the genome.
• A given RFLP marker frequently occurs in numerous variants in a population (hence, polymorphisms)
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A DNA fingerprint is a specific pattern of RFLP
bands
STRs – variations innumber of tandem repeated base sequences found in satellite DNA.
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Human DNA Fingerprinting
Father and four children.Which lane contains the DNA of the father?
Which child is least likely to be the biological offspring of these parents?
Lane 3 is the only lane that shares one band with each of the other lanes.
Child 2
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The Human Genome Project
• Goals:1. Determine nucleotide sequence for entire human genome (3 X 109 bps).
2. Map the location of every gene on each chromosome.3. Compare to genomes of other organisms.
• Fundamental questions:1. How are genomes organized?2. How is gene expression controlled?3. How are cell growth and differentiation under genetic control?4. How does evolution occur?
• Scientists have discovered specific genes responsible for several genetic disorders:
1. Cystic fibrosis2. Duchenne muscular dystrophy3. Colon cancer
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Human Genome Project• International consortium of 20 groups of researchers. • Included mapping of important research organisms –
E. coli, yeast, C.elegans (nematode), and mouse.• Three stages:
1. Genetic (linkage) mapping – develop a map of several thousand genetic markers (genes, RFLPs, STRs)2. Physical mapping: ordering DNA fragments using restriction fragments (overlap)3. DNA sequencing using clones of short DNA fragments and,
later, DNA sequencing machines.
• Later, J.C. Ventra (Celera Genomics) used powerful computer programs to order the large number of random short sequences cut from the whole genome.
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Studying Gene Expression
DNA Microarray Assays
Red – genes expressed in sample A.Green – genes expressed insample B.Yellow – genes expressed equally in both samples.
Used to determine which genesare expressed in response to aspecific treatment or disease.Also – tissue specific genes.
DNA microarray - full set of 1,000sof sequences
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Determining Gene Function
• In vitro mutagenesis:
1. Changes are made to a gene2. Altered gene returned to cell3. Monitor changes in physiology
or developmental patterns
• RNA interference (RNAi):
- Synthetic double-stranded RNA molecules that match a gene sequence trigger the breakdown of that gene’s mRNA.
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Practical Applications of DNA Technology
• Medicine:-Diagnosis of infectious diseases and genetic disorders
-Human gene therapy – currently aimed at fighting heart disease and cancer-Pharmaceutical products – growth hormones, insulin, vaccines
• Forensics:-DNA fingerprints
-Use simple tandom repeats (STRs) found in satellite DNA-Five small regions of the genome known to vary widely
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Practical Applications of DNA Technology (cont.)
• Environmental – genetically engineered organisms:– Able to extract heavy metals– Sewage treatment– Research – engineering organisms to degrade chlorinated
hydrocarbons and other toxic chemicals- Bioremediation – cleaning up oil spills and waste dumps
• Agricultural: - Transgenic organisms
-increased productivity-pest resistance, disease resistance-”pharm” animals-”golden rice” – enriched with beta-carotene
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Safety and Ethical Questions