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46 Cards in this Set
- Front
- Back
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Recombinant DNA Technology
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a set of molecular techniques for locating, isolating, altering, and studying DNA segments
-combine DNA from 2 distinct sources |
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Molecular Techniques Require
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1. Isolation
2. Recombination 3. Amplification |
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Restriction Enzymes
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recognize and make double-stranded cuts in DNA at specific nucleotide, produced naturally by bacteria, used in defense of viruses, it modifies the recognition sequence for protection
-3 different types: mainly use type II (III)-cuts dna outside recognition sequence |
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Recognition Sequences
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most are palindromic, recognized by restriction enzymes, all type II restriction enzymes recognize type II
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Cohesive Ends/Sticky Ends
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complementary to eachother and can spontaneously pair to connect the fragments, once together the DNA can be joined together permanently by DNA ligase
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Gel Electrophoresis
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used to separate DNA molecules based on their size and electrical charge
-because of the P negative charge, the DNA fragments migrate to the + end of the gel |
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Autoradiography
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a piece of X-ray film is placed on top of the gel, used to detect radioactively labeled DNA
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Southern Blotting and Probes
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probe: bases on a probe will pair only with the bases on a complementary sequence, and can be used to locate a specific gene or other DNA sequence
-SB: after fragments denatured and transferred to a permanent solid medium, placed in a hybridization bottle, gently rotated |
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Northern Blotting
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RNA can be transferred from a gel to a solid support
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Western Blotting
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protein transfer from a gel to a membrane
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Gene Cloning
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identical copies of the original piece of DNA are produced
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Cloning Vector
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stable, replicating DNA molecule to which a foreign DNA fragment can be attached for introduction into a cell
-has an origin of replication -selectable markers -one or more unique restriction sites |
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Plasmid Vector
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able to replicate independently of the bacterial chromosome, for inserting a gene into a plasmid, vector is to cut the foreign DNA and the plasmid with the same restriction enzyme, DNA and plasmid mixed together
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Linkers
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small synthetic DNA fragments that contain or or more restriction sites, can attach to the ends of DNA and then cut by restriction enzyme, generating sticky ends
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Transformation
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capacity of bacterial cells to take up DNA from the external environment
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Selectable Markers
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lacZ, in the absence of an inserted fragment lacz is active and produces B-galactosidase
-when foreign DNA is inserted into the restriction site, it disrupts the lacz and B-galactosidase is not produced |
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Screen Bacteria Recombinant Plasmids
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-foreign DNA is inserted into the middle of the lacZ gene
-Bacteria that are lacZ- are tranformed by the plasmids -Bacteria with an intact plasmid produce B-galactosidase, which cleves X-gal and makes the colonies blue -bacteria with a recombinant plasmid do not synthesize B-galactosidase their colonies remain white(recombinant plasmid) -bacteria without a plasmid will not grow |
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Cosmids
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plasmids that are packaged into empty viral protein coats and transferred to bacteria by viral infection, can carry more than twice as much foreign DNA as can a phage vector
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Bacterial Artificial Chromosomes
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are vectors originally constructed from the F plasmid, a special plasmid that controls mating and the transfer of genetic material in some bacteria
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Expression Vector
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contains sequences required for transcription and translation in bacterial cells
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Yeast Artificial Chromosome
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is a DNA molecule that has a yeast origin of replication, a pair of telomeres and a centromere, useful because they can carry more DNA fragments (600 kb-1000kb)
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Ti Plasmid
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part of which is transferred to a plan cell when A.tumefaciens infects a plant, integrates into one of the plant chromosome, transcribed and translated to produce several enzymes that help support the bacterium
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Polymerase Chain Reaction
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allows DNA fragments to be amplified a billionfold within just a few hours
-replication catalyzed by a DNA polymerase 1. single stranded DNA template from which a new DNA strand can be copied 2. primer with a 3 OH group wo which new nucleotides can be added |
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Process of PCR
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DNA heated to 90-100 C to separate the two strands, cooled quickly to 30-65 C to allow short single-strand primers to anneal to their complementary sequences, heated to 60-70C DNA polymerase synthesizes new DNA strands, creating 2 new double-stranded DNA molecules, the entire cycle is repeated, each time the amount of target DNA is doubled
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Reverse-Transcription PCR
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can also be used to amplify sequences corresponding to RNA, have to convery RNA to corresponding DNA (cDNA)
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Limitations of PCR
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1. requires prior knowledge of at elast part of the sequence of the target DNA to allow the construction of the primers
2. contamination of a significant problem 3. accuracy, taq polymerase does not have the capacity to proofread 4. the size of the fragments that can be amplified |
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Real-time PCR
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can be used to quantatively determine the amount of starting nucleic acid, the amount of DNA amplified is measured as the reaction proceeds
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DNA Library
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a collection of clones containing all the DNA fragments from one source
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cDNA Library
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library consisting only of those DNA sequences that are trascribed into mRNA, all DNA in this library is complementary to mRNA
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creating cdna library
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-special comumn contains short oligo(dt) chains linked to cellulose
-total cellular RNA is isolated from cells and passed through the column -the poly(a)tails of mRNA pair with the o(dt) chains and the mRNA is retained in the column -the rest of the RNA passes through -mRNA is washed from the column by adding a buffer that breaks the H bonds -leaves only mRNA and poly(a) tails -o(dt) primers anneal to the poly a tails of the mRNA and provide 3 OH groups for DNA synthesis -reverse transcriptase synthesizes a DNA strand by using the mRNA as a template -the RNA-DNA hybrid molecule is briefly treated with RNase, which partly digests the RNA strand -DNA polymerase synthesize the 2nd DNA strand by using the short undigested RNA pieces as primers -the nicks in the sugar-phosphate backbone are sealed by DNA ligase |
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Screen a gene library
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-disc of nitrocellulose or other membrane is laid on top of the bacterial colonies
-few cells from each colony adhere to the nitrocellulose filter -cells are disrupted and their DNA is denatured and fixed to the filter -labeled probe hybridizes with any complementary DNA -detects the presence of the probe -comparison of the membrane with the master plate reveals which bacterial colonies have the DNA of interest |
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In Situ Hybridization
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DNA probes are used to determine the cellular or chromosomal location of a gene or its product
-requires cells to be fixed and chromosomes be spread on a microscope slide and denatured -can carry fluorescant dyes that can be seen directly with the microscope |
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Positional Cloning
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to isolate genes on the basis of their position on a gene map
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Chromosome Walking
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by making probes complementary to areas of overlap between cloned fragments in a genomic library, we can connect a gene of interest to a previously mapped, linked gene-used to locate a gene of interest
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Chromosome Jumping
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used to locate distantly linked clones
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Restriction Fragment Length Polymorphisms
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one group of markers which are variations in the patterns of fragments produced when DNA molecules are cut with the same restriction enzyme
-Huntingtons disease, one has two sites, the other only has one, therefore produce two different patters, hetero would produce both sets of bands |
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DNA Sequencing
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quickly determines the sequence of bases in DNA, requires dNTP and ddNTP(identical to dNTP except that they lack a 3OH group for synthesis, after it is incorporated, no more nucleotides can be added)
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Dideoxy method of DNA
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based on the termination of DNA synthesis
-each of four reations contains single-stranded target DNA to be sequences -a primer is added -all four deoxyribonucleoside triphosphates, DNA polymerase added -and one type of ddATP, ddCTP, ddGTP, ddTTP -nucleotides are added to the 3' end of the primer, with the target DNA being used as a template -when dideoxy is added into the growing chain synthesis terminates because it lacks a 3'OH -terminated on different strands, generates a set of DNA fragments of various lengths, each ending in a dideoxy with the same base -fragments produced in each reaction are separated by gel electrophoresis -read from gel, starting from the bottom -the sequence obtained is the complement of the original template strand |
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DNA Fingerprinting
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the use of DNA sequences to identify individual persons, most use mocrosatelites or short tendem repeats
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DNA Fingerprinting process
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-DNA samples are collected and subjected to PCR
-the elngth of the DNA fragment produced by PCR depends on the number of copies of the microsatellite sequence -the fragments are separated by gel electrophoresis. different-size fragments appear as different bands -multiple microsatellite loci produce multiple bands on the gel |
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Forward Genetics
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begins with a phenotype and proceeds to a gene that encodes the phenotype
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Reverse Genetics
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begin with a genotype-a DNA sequence-and proceed to the phenotype by altering the sequence or inhibiting its expression
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Site-Directed Mutagenesis
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mutations are induced at specific locations, restriction enzymes cut out a short sequence of nucleotides that is then replaced by a synthetic mutated DNA sequence
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Transgenic Animals
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organism that has been permanently altered by the addition of a DNA sequence to its genome
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Knockout Mice
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useful variant of the transgenic approach, produce mice in which a normal gene has been not just mutated, but fully disabled
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RNAi
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temporarily turn off gene and observe phenotype
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