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Piece of DNA that can replicate automously within a bacteria, carry foreign piece of DNA
Vectors
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where in the plasmid is the DNA fragment inserted? how is this region cut?
MCS (mutiple cloning site), use restriction endonuclease digestion
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Name 3 main functions of vectors
Replication, sequencing, protein expression
- for Rep- has ORI
- for Seq- has primer
- for Exp- has promoter
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Sequence a piece of DNA by dideoxynucleotide (when incorporated, terminates the DNA strand synthesis)
Sanger (dideoxy) sequencing
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Advancement of analyzing the data of sanger sequencing
- fluorescent label - allow all four kinds of labeled strands to be loaded into one gel
- machine- automated by laser and detector
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websites for finding hereditary disease? for finding gene sequences?
OMIM, GenBank
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how many base pairs are there in human genome
3.2 billion!
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Technique used for detecting RFLPs
- southern blot
- (restriction digestione, hybridization, X-ray film)
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Used to detect single base mutation
ASO (allele specific oligonucleotide) probes
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this pencure that pcr is working properly ( prevent diagnostic call during scres up)
internal control
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Severity of this disease is determined by the number of triple repeat? what is the code?
Huntington's disease, CAG
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what technique is used to diagnose Huntington's disease
Allele specific PCR
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State two techniques used for chromosomal abnormalities. How are hese two different?
Standard Karyotype analysis, FISH. Both detects duplication, deletion, and rearrangement of chromosomes. However, Karyotype is for major abnormalities of chromosomes. FISH is for microdeletion.
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what techinique is used for diagnosing Down syndrome (trisomy 21)
aneuploid screen test, standard karyotype
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What technique is used for diagnosing DiGeorge's syndrome
FISH (DiGeorge is due to microdeletion of chromosome 22)
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what phase chromosomes are used for FISH
metaphase chromosomes (highly coiled)
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Techinique used for diagnosing steroid sulfatase deficiency
FISH
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technique used for ID genes differently expressed(produce RNA) in two tissues
DNA microarray
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technique used for camparting protein species in two tissues
2D protein gel electrophoresis
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in DNA microarray, what is bound to to the microarray
cDNA binds to the microarray ( this cDNA is derived from mRNA in tissues and labeled before being applied to the microarray)
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Distinguish Southern, Northern, Western
- Southern-DNA
- Northern-mRNA
- Western-protein
(snow drop)
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method used for first dimension of 2D gel
isoelectric focusing
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labeling method used after isoelectric focusing
fluorescent labeling
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mice used to human disease model
- KO mice
- (transfect cell with vector containing targeting construct, chemical elimination of cells not properly transfected, ES cell injection to balstocysts)
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deletion of this gene cause limb trunction and failure of anterior and posterior neuropore
sp8 gene
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technique for expressing recombinant protein in hosts
expression vectors
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technique used for producing large quantities of human protein in milk
also used when you can't produce protein from bacteria
transgenic ruminants
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three examples of proteins produced from transgenic ruminants
insulin, anit-thrombin (aniti clotting factor), antibodies
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