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Genetics Chapter 10
Terms in this set (55)
how many ways are there to amplify a gene of interest
a term that describes the collective techniques for obtaining, amplifying, and manipulating specific DNA fragments
the application of DNA technology to specific biological, medical, or agricultural problems, is
now a well-established branch of technology
is the ultimate extension of the technology to the global analysis of the nucleic acids present in a nucleus, a cell, an organism, or a group of related species
a specially designed plasmid or bacterial virus that will "carry" and amplify the gene of interest
when DNA molecules are cut and inserted into a chromosome vector they form
A section of DNA that has been inserted into
a vector molecule, such as a plasmid or a phage, and then replicated to produce many copies.
A section of DNA that has been inserted into a vector molecule, such as a plasmid or a phage, and then replicated to produce many copies.
in the test tube using the polymerasechain- reaction technique. Both methods employ the basic principles of molecular biology: the ability of specific proteins (red and green) to bind to DNA and the ability of complementary single-stranded nucleic acid segments to hybridize together
polymerase chain reaction
in vitro method for amplifying a specific DNA segment that uses two primers that hybridize to opposite ends of the segment in opposite polarity and, over successive cycles, prime exponential replication of that segment only.
These enzymes cut at specific DNA sequences, called restriction sites, and this property is one of the key features that make restriction enzymes suitable for DNA manipulation (examples of endonucleases)
A DNA fragment resulting from cutting DNA with a restriction enzyme
both strands have the same nucleotide sequence but in antiparallel orientation
Combining complementary single-strands so that they can base-pair is called
what is a powerful technique that is routinely used to isolate specific genes or DNA fragments when there is prior knowledge of the sequence to be amplified.
Complementary DNA (cDNA)
is a DNA version of an mRNA molecule. Researchers use cDNA rather than mRNA itself because RNAs are inherently less stable than DNA
double stranded cDNA is synthesized from
backbones can be covalently sealed by the addition of the enzyme _____________, which creates phosphodiester linkages at the junctions
what produces a hairpin loop that works as the primer for DNA polymerase.
double stranded cDNA
transcription occurs in the cell and creates and mRNA, the introns are then spliced, the oligo primers are added and anneal, reverse transcriptase occurs copying mRNA to cDNA, hairpin loop from cDNA is created as mRNA is removed,( RNA is removed by either RNAseH or with basic solution) DNA polymerase copies cDNA strand, nick haipin creating
To make recombinant DNA molecules containing donor
genomic DNA fragments, both donor and vector DNAs are digested by a restriction
enzyme that produces the same complementary
To make recombinant DNA molecules containing donor genomic DNA fragments, both donor and vector DNAs are digested by a restriction enzyme that produces the same complementary
vehicles that contain the gene or DNA of interest that can amplify it away from the native genome
Characteristics of Vectors
1.Origin of replication for host cell
2.Unique restriction enzyme sites for insertion so do not cut other regions of the vector
3.Selection for inserts such as the inactivation of lacZ (see slide 11) is ideal
you choose these based on
1.Desired size of insert
2.Copy number of insert
1 to 10 kb insert; high copy number e.g. pGLO
10-18 kb insert; protected in phage coat; high yield
35- 45 kb insert; single copy, so low or no recombination
Most of human genome covered in ______ and gap coverage was done with YACs.
what sites allow fosmids to be packaged in phage heads in vitro.
engineered hybrids of λ phage DNA and bacterial F plasmid DNA
what delivers recombinant DNA into bacterial cells via transformation
what delivers recombinant DNA into bacterial cells via transduction
what delivers recombinant DNA into bacterial cells via transduciton
a collection of fragments in vectors housed in bacteria, phage, or yeast clones
100 - 200 kb insert; single copy
100 - 10,000 kb insert; single copy; grown in E. coli, then insert added and transformed into yeast.
types of Libraries
1.Genomic (usually refers to nuclear)—clone 5X genome size to get full coverage
2.Mitochondrial or chloroplast genomic
3.cDNA -- the complementary copy that represents mRNAs (will differ under different conditions e.g. tissue type, time of development, exposure to different environmental paramerters)
Ways to find you clone of interest in a ____________
1.Hybridization of complementary labeled probe
2.Clone in expression vector and use antibody to detect your protein of interest
3.Use amino acid sequence to engineer a set of oligos
4.Functional rescue—see if clone rescues mutant phenotype by transforming mutant lines with various clones
DNA on gel
RNA on gel
protein on gel
use the genetic position to isolate the gene underlying the trait
use the sequence of the nearby landmark as a probe to identify a second set of clones that overlaps the marker clone containing the landmark but extends out from it in one of two directions
recombinant DNA technology = biotechnology (application)
cutting and pasting DNA and inserting into new organisms
is a protein made by an animal's immune
system that binds with high affinity to a given molecule.
can tell us about our clones and our clones can tell us something about genomes by amplifying regions in the "smear"
insert can replace endogenous gene or insert elsewhere, aslo called side effects when engineering animals and plants and humans,
what can cause problems because they can mutate the region in which they insert or can be expressed in the wrong place at the wrong time because of the region in which they were inserted.
what can be introduced into a eukaryotic cell by a variety of techniques, including transformation, injection, bacterial or viral infection, and bombardment with DNA-coated tungsten or gold particles using a gene gun
causes the dNTP strand to stop as it blocks DNA synthesis. lacks the 3′-hydroxyl group as well
as the 2′-hydroxyl group, which is also absent in a deoxynucleotide. DNA polymerase tries to catalyze the reaction to the 3' end, but because there is no hydroxly group at the 3' end at the dNTP then it can not bind thus stopping the addition
Genetically modified organisms
DNA can now be introduced from other
species of plants, animals, or even bacteria, producing
works well in tobacco, brassica and relatives. A bacterial plasmid that has been inserted in root genomes naturally for hundreds of years.
Random insertion can lead to _____________ and multiples can lead to scrambling
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