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57 Cards in this Set
- Front
- Back
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• Recombinant DNA
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o DNA in which nucleotide sequences from two different sources are combined in vitro into the same DNA molecule
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• Genetic engineering
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o Direct manipulation of genes for practical purposes
o Recombinant DNA is important to genetic engineering |
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• Biotechnology
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manipulation of organisms or their components to make useful products
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• Gene cloning
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methods for preparing well defined gene sized pieces of DNA in multiple identical copies
• E Coli is often used to clone DNA o Foreign DNA is inserted into a plasmid from the bacteria o The plasmid is now a recombinant DNA molecule o Recombinant plasmid is inserted into bacteria, making the bacteria recombinant bacteria (a clone) o Bacteria copies itself and thus copies the plasmid with the gene • Use cloned genes to make many copies of a gene and to produce protein product |
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• Restriction enzymes
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enzymes that cut DNA molecules at a limited number of specific locations
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• Restriction site
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short DNA sequences that tells a restriction enzyme where to cut
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• Restriction fragments
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o Since restriction sites are so short, there are a lot of restriction sites that occur by chance in a DNA molecule
o Restriction enzymes cut at every restriction site, yielding lots of restriction fragments |
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• Sticky end
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when restriction enzymes cut DNA, the ends of the DNA fragment are single stranded
o Sticky ends can stick to other complementary sticky ends • If one sticky end has the nucleotide sequence AAA, it can bond to another sticky end with the sequence TTT • Then the two fragments will join together |
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• DNA ligase
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joins restriction fragments together by bonding sticky ends together
o Forms covalent bonds that seals the sugar phosphate backbones of the joined DNA fragments • When two DNA fragments join together by stick ends, they form a recombinant DNA fragment |
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• Cloning vector
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the original plasmid- the DNA molecule that carries foreign DNA into a cell
• Bacterial plasmids make good cloning vectors because they’re easily manipulated and bacterial cells reproduce quickly |
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• How to clone a eukaryotic gene with a bacterial plasmid
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o Isolate the bacterial plasmid from the bacteria
o Isolate the human DNA you want to study o Put the amp gene and the lacZ gene into the bacterial plasmid o Digest the bacterial plasmid and the human DNA with a restriction enzyme that produces sticky ends • Enzyme cuts the plasmid DNA at the lacZ gene restriction site • Enzyme cuts the human DNA at multiple sites o Mix human DNA fragments with the cut bacterial plasmid DNA • Complementary sticky ends join together o Add DNA ligase to join the human and plasmid DNA together o Mix the recombinant human and bacterial DNA with bacteria that have a mutation in their own lacZ gene so they can’t hydrolyze lactose o Bacteria suck up the recombinant DNA through transformation o Put all the bacteria on agar with ampicillin and imitation lactose • As the cloned bacteria divides it reproduces the DNA of interest • But you still don’t know which bacteria has the right gene you want- you will have cloned many different human DNA fragments b/c there are so many restriction sites |
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what does the amp gene do
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• Amp makes it resistant to ampicillin
o The bacterium that soak up the right recombinant plasmid will survive b/c they’re resistant to the ampicillin |
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what does the lacZ gene do?
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• lacZ encodes beta-galactosidase, hydrolyzes the sugar Lactose, and contains the restriction site
o bacterium that soak up the right recombinant plasmid will be white b/c they can’t hydrolyze Lactose b/c their lacZ gene was disrupted by the restriction enzyme- they can’t make beta galactosidase o Bacterium that don’t soak up the right recombinant plasmid will be blue b/c they will have an intact lacZ gene and they can hydrolyze lactose and make beta-galactosidase |
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• Colony
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mass of bacteria on the agar
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o Nucleic acid hybridization
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• Identifying a gene through detecting its ability to base pair with a complementary sequence of another single stranded nucleic acid molecule (a nucleic acid probe- could be DNA or RNA)
• Nucleic acid probes are labeled with a radioactive isotope so they can be seen and identified |
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o Denaturation
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→ splitting DNA into two single strands- can be done through heat or chemicals
• You need to denature DNA in order to use a nucleic acid probe |
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• Genomic library
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the complete set of plasmid clones that each carry a copy of a particular segment from the initial genome
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• cDNA
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complementary DNA
o reverse transcriptase makes single stranded DNA transcripts of mRNA molecules o a second DNA strand which is complementary to the first is synthesized by DNA polymerase o the DNA is now double stranded (called cDNA) o cDNA is modified by adding restriction enzyme recognition sequences to each end o cDNA is inserted into vector DNA o since the mRNAs that made the cDNAs are a mixture of all the mRNA molecules from the cell they came from, you have a bunch of cDNAs that have different genes in them→ a cDNA library |
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genomic libraries
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the complete set of plasmid clones that each carry a copy of a particular segment from the initial genome
o Genomic libraries are useful • if you don’t know what cell type your gene is expressed in • if you can’t obtain the cell type needed to express your gene • if you want to study introns (cDNA libraries come from mRNAs which don’t contain introns) |
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cDNA libraries
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o cDNA libraries are useful
• for studying genes responsible for the specialized functions of a particular cell type • for studying changes in pattern of gene expression during a cell’s development by making cDNA from cells of the same type at different times in the life of an organism |
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• Expression vector
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• sometimes it’s hard expressing a eukaryotic gene in a prokaryotic cell b/c the systems are so different
→ cloning vector that contains a highly active prokaryotic promoter near the restriction site where the eukaryotic gene can be inserted o Bacterial host cell will recognize the promoter and express the foreign gene that’s linked to the promoter o Allows expression of eukaryotic genes in bacterial cells |
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• Advantages of using yeast cells/single celled fungi as host cells
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• it’s sometimes hard to grow a eukaryotic gene in a prokaryotic cell
• It’s hard to express a eukaryotic gene in a prokaryotic cell b/c prokaryotic cells can’t splice out the introns in eukaryotic DNA some proteins don’t function unless they’re modified after translation (bacteria can’t carry out modifications) o yeast are easy to grow o They have plasmids (most eukaryotes don’t have plasmids) |
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• Yeast Artificial Chromosomes (YACs)
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o Have an origin for DNA replication, centromere, two telomeres
o Combine the essentials of a eukaryotic chromosome with foreign DNA o Act as vectors→ multiply while the yeast divides o Can carry longer DNA segments than plasmid vectors |
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• Electroporation
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o Electrical pulse is applied to the cells, this punctures temporary holes in their membranes
o DNA can go through the holes o Method of getting recombinant DNA into eukaryotic cells • Scientists can also inject recombinant DNA into eukaryotic cells with microscopic needles |
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• Polymerase Chain Reaction (PCR)
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o Makes copies of (amplifies) the DNA you want in a test tube
o Faster than cloning DNA in cells o Denature the DNA with heat o Single stranded DNA primers bond to the complementary ends of the target DNA • The primers ensure that only the target DNA gets copied- very specific o A heat stable DNA polymerase extends the primers in the 5→3 direction and copies the DNA |
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• Restriction fragment analysis
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detects differences in the nucleotide sequences of DNA molecules
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• Gel electrophoresis
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o Uses a gel to separate nucleic acids or proteins according to size, electrical charge, and other physical properties
o Uses electrical charge to pull DNA towards the positive end and separate DNA fragments by size • Different DNA nucleotide sequences will yield different banding patterns when you perform gel electrophoresis on it b/c the fragments will be different sizes b/c the enzymes will cut at different places b/c the coding is different o So restriction fragment analysis is useful for comparing two different DNA molecules |
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• Southern blotting
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o Combines gel electrophoresis and nucleic acid hybridization
o Used to detect specific nucleotide sequences within a DNA sample o Useful for comparing restriction fragments o 1) Digest DNA samples with restriction enzyme o 2) place restriction fragments into a gel and perform gel electrophoresis o 3) Transform the DNA from the gel onto a piece of nitrocellulose paper o 4) mix the nitrocellulose paper (which contains the DNA) with a radioactive probe that binds to the DNA fragments that contain the gene you want o 5) the radioactive probe allows you to see the fragments that contain the gene you want o reveals if a particular sequence is present in a sample of DNA and also reveals the size of the restriction fragments |
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• restriction fragment length polymorphisms (RFLPs)
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• scientists discovered that noncoding DNA demonstrates differences in coding also
o different restriction fragment patterns that come from differences in the restriction sites on homologous chromosomes o a type of sequence difference in noncoding DNA o RFLP’s can serve as genetic markers for a particular location in the genome o Detected and analyzed with southern blotting o RFLPs are inherited in a mendelian fashion o The frequency with which two RFLP markers are inherited together indicates the closeness of the two loci on a chromosome o Used as markers for mapping the human genome |
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• Human Genome Project- 1990 - 2003
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o Goal was to map out the human genome
o Three stages→ genetic linkage mapping, physical mapping, DNA sequencing |
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• Cytogenic map
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chromosome banding pattern and location of specific genes by fluorescence in situ hybridization (FISH)
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• Genetic (linkage) mapping
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o Ordering of genetic markers such as RFLPs, simple sequence DNA, and other polymorphisms
o Construct a linkage map of several thousand genetic markers spaced throughout each of the chromosomes o Order and distance between the markers are based on recombination frequencies o Markers can be genes or any other identifiable sequence of DNA like RFLPs o Linkage maps serve as a framework for organizing more detailed maps of particular regions |
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• Physical mapping
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o Ordering of large overlapping fragments cloned in YAC and BAC vectors, followed by ordering of smaller fragments cloned in phage and plasmid vectors
o Distances between markers are expressed in a physical measure (# of base pairs along the DNA) |
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• DNA sequencing
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o Determination of nucleotide sequence of each small fragment and assembly of the partial sequences into the complete genome sequence
o Determine the complete nucleotide sequence of each chromosome o Dideoxyribonucleotide chain termination method |
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o Dideoxyribonucleotide chain termination method
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• Machine that determines nucleotide sequence
• Denature the DNA you want to sequence • Attach a primer to one end of the DNA and continue synthesizing a complementary strand until you insert a dideoxyribonucleotide which prevents further elongation • Dideoxyribonucleotide→ modified nucleotide tagged with a fluorescent label • Eventually you have a set of labeled strands of various lengths with the color of the tag representing the last nucleotide in the sequence • Run each fragment through a polyacrylamide gel so shorter strands travel further than longer strands • Fluorescence detector senses the color of each fluorescent tag • Strands differing by as little as one nucleotide in length can be distinguished • The color of the fluorescent tag on each strand indicates the identify of the nucleotide at its end, when you put all the fragments together you can sequence the entire DNA |
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o J Craig Venter developed another way to sequence genomes
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• Cut DNA from many copies of an entire chromosome into overlapping fragments
• Clone the fragments in phage or plasmid vectors • Sequence each fragment • Order the sequences into one overall sequence with computer software • Venter developed Celera Genomics which sequenced 90% of the human genome |
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• Genomics
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study of whole sets of genes and their interactions
o Geneticists can study genes directly without having to infer genotype from phenotype o But now you have to determine the phenotype from the genotype |
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• Indicators of protein coding genes
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o Sequences for transcriptional and translational start/stop signals
o RNA splicing sites o ESTs (expressed sequence tags)→ sequences similar to those present in known genes o If you find any of these in a gene it’s probably a protein coding gene |
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• Genome size does not correlate with biological complexity among eukaryotes
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o Some flowers have more genes than humans
o There’s lots of noncoding DNA in human genome |
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• So what makes some organisms like vertebrates more complex than others?
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o Genome size is not correlated with biological complexity
o Gene expression is regulated in more subtle and complicated ways in vertebrates o Vertebrate genomes can do more with less genes b/c of alternative splicing of RNA transcripts • Alternative splicing generates more than one functional protein from a single gene o Polypeptide diversity o Larger number of possible interactions between gene products that result from greater polypeptide diversity |
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• In vitro mutagenesis
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o Mutations are introduced into the sequence of a cloned gene
o If the mutations alter or destroy the function of the gene product the phenotype of the mutant cell can help reveal the function of the missing normal protein |
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• RNA interference (RNAi)
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o Double stranded RNA molecules block or break down a gene’s mRNA
o Used to prevent gene expression |
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Determining Gene Function
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• Disable the gene and observe the consequences in the cell/organism
• In vitro mutagenesis • RNA interference (RNAi) |
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• DNA microarray assays
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o Used to test thousands of genes at once to determine coordinated gene expression
o Isolate mRNA from a tissue sample o Make cDNA by reverse transcription using fluorescently labeled nucleotides o Apply the cDNA mixture to a microarray (microscopic slide that holds copies of single stranded DNA fragments from an organism’s genes, a different gene in each spot) o The cDNA hybridizes with any complementary DNA on the microarray o Rinse off excess cDNA o Scan the microarray for fluorescence o Each fluorescent spot represents a gene expressed in the tissue sample |
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Comparing genomes of different species
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• Comparing genome sequences from different species helps to determine evolutionary relationships between species
• Genomes of organisms like yeast and fruit flies help us understand human genome • Comparing genomes of closely related species is helpful b/c their genomes are likely to be organized similarly • Since closely related species have a small number of gene differences, it’s easy to correlate phenotypic differences with genetic differences |
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• Proteomics
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o Studying full protein sets encoded by genomes
o It’s important to study proteins b/c it’s the proteins that carry out the cell’s activities o Also the number of proteins exceeds the number of genes |
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• Single Nucleotide Polymorphisms (SNPs)
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o Single base pair variations in the genome
o Most human diversity is in the form of SNPs o You and another person are 99.9% identical |
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Medical applications
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• DNA technology helps identify gene mutations that create genetic diseases
• DNA technology increases understanding of non genetic diseases like aids and arthritis • Helps to diagnose diseases • Use PCR and labeled nucleic acid probes to track down certain pathogens • Can diagnose genetic disorders using PCR and primers corresponding to cloned disease genes and then sequencing the amplified product to look for the disease causing mutation • Detection of a RFLP marker that’s near a disease gene can detect the disease gene gene therapy |
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• Gene therapy
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alteration of an afflicted individual’s genes
• Helpful for treating disorders traceable to a single defective gene o bone marrow cells (which include stem cells that become the blood and immune system) • Put normal allele into retrovirus • Let virus infect patient’s bone marrow cells • Viral DNA inserts normal allele into bone marrow chromosome • Inject engineered cells into patient • Now the bone marrow will produce the normal protein |
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• HGH (human growth hormone) and human insulin
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first pharmaceutical products manufactured through cloning
o HGH-→ to treat dwarfism o Insulin→ diabetes |
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• TPA (tissue plasminogen activator)
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o Treats people after heart attacks to stop clotting and reduce risk of subsequent attacks
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• DNA fingerprint
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specific pattern of bands unique to each individual→ southern blotting can reveal the DNA fingerprint
• DNA fingerprinting can establish paternity, the criminal at the scene of a crime o There’s a very low chance that two people will share the same DNA fingerprint • Southern blotting can detect DNA at the scene of a crime with a small amount of blood sample • RFLPs and variations in the lengths of certain repeated base sequences are used as markers for DNA fingerprinting |
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• STR’s (simple tandem repeats)
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o Repetitive sequences of DNA that are unique to each person
o Helps identify DNA fingerprint o PCR is often used to amplify STR markers |
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Environmental Cleanup
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• Scientists engineer organisms with the ability to transform chemicals for environmental clean up
o Ex: some bacteria can extract and process metals from the environment o Engineered organisms are used to clean up toxic wastes o Some bacteria have been engineered to clean up oil spills |
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• Transgenic animal
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when scientists introduce a gene from one animal into the genome of another animal
o Take the egg from the female and fertilize it in vitro o Clone the desired gene from another organism o Inject the cloned gene DNA into the fertilized egg o Transgene→ foreign DNA o If the embryo develops successfully, it will be the product of three parents (the third parent being from another species) o Why make transgenic animals? • Sheep with better wool, pig with leaner meat, cow that matures in shorter amount of time • You can implant a gene from a human into an animal and make the animal produce a desired protein for humans (like blood clotting factor) |
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• Ti Plasmid
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• Can engineer plants to resist spoilage and disease, delayed ripening, increased nutritional value
most commonly used vector for introducing new genes into a plant cell o Integrates a segment of its DNA called T DNA into the DNA of its host plant cell o Steps… • Restriction enzyme cuts the T DNA • Gene of interest is placed in the T DNA where the cut was made • Now you have a recombinant Ti Plasmid with the foreign gene in it • Put the recombinant plasmid inside the plant- the Ti Plasmid will integrate its T DNA (and the foreign DNA) into the plant’s DNA |
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• Genetically Modified Organisms (GM)
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o Pose possible hazards when used in food
o GM organism is one that has artificially acquired genes from the same or another species o Salmon are genetically modified to grow faster o GM plants could be potentially hazardous • Transgenic plants could pass their genes to other wild plants and genetically modify them |
