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30 Cards in this Set

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Approval process for clinical medicines?
Indentification of a target for therapeutic intervention, pilot scale production, pre-clinical trials, clinical evaluation, Phase I; safety
Phase II; safety and efficacy
Phase III; controlled safety and efficacy.
FDA approval
Takes 7-12 years
When did Recombinant DNA technology begin?
1970s
What was the purpose of Recombinant DNA technology?
To manipulate DNA for study
What are the goals/benefits of Recombinant DNA technology?
-To decipher genomes
-Create treatments
-Overcome mutations
-Understand and regulate gene expression
-Synthesize proteins
-GMOs
Basic tools-restriction enzyme analysis?
Cuts DNA into certain segments
Blotting techniques include:
Southern (blotting DNA), Western blotting (protiens), and Northern blotting (RNA).
DNA sequencing
Determining a precise nucleotide sequence
Synthesis
of nucleic acids
PCR as a tool?
Amplification of target DNA sequences, allows one molecule to be amplified for characterization and manipulation. Computer-bioinformatics
What is purpose PCR?
The purpose of polymerase chain reaction (PCR) is to produce a huge number of copies of a gene; to amplify, amplify, and amplify.
How does PCR work, reaction components?
-Target DNA
-Primers
-dNTPs
-DNA polymerase
-MgCL
-Buffer components
PCR--Primers
Primers are made that will complement the denatured target DNA. Avoid homologous primers and primer dimers. Consider secondary structure of primers, primers are 18-30nt in length. Higher specificity, generally the longer primer.
Cyle of PCR
94 C-Denaturation; denatures DNA, target DNA and primers are now single stranded.
58 C-Annealing; primers drop down onto template DNA and anneal
72 C-Elongation; at the ideal temperature of the polymerase; polymerase replicates template
Restriction enzymes also called?
endonucleases
What is the biological role of restriction enzymes?
To recognize foreign DNA and cleave it.
When DNA is cut by restriction enzymes it is said to be?
digested. A restriction enzyme digestion
DNA analysis
Fluorescent tag is attached to each oligonucleotide priming fragment, colors show based sequence based on how they fluoresce. No radioactivity used.
DNA vectors
Joined to a DNA fragment of interest, replicate autonomously in host, plasmids, cleave with RE, forms staggered ends, complementary, called sticky ends. The single stranded ends are then complementary to the plasmid, anneal with DNA ligase, DNA ligase catalyzes the formation of phopshodiester bonds at breaks in the strand.
What are the basic steps in cloning?
1. DNA isolation
2. Restriction enzyme digest, obtain gene of interest
3. Isolate fragment
4. Ligation of vector and fragment
5. Transformation-Transformation of genetic information from one systme to another by means of DNA, electroporation so replication can happen in a host with machinery
6. Selection and identification
Joining the DNA
Cohesive end method utilizes a short synthesized DNA linker. First the linker is joined to the ends of a fragment or vector. Functions to add cohesive ends corresponding to a particular RE, etc.
Plasmids as vectors
Modified to enhance the delivery of recombinant DNA molecules into bacteria and to select for bacteria haboring these vectors.
Plasmids as vectors shape?
Circular DNA, 2-several hundred Kb
Plasmids as vectors
Antibiotic resistance genes, produce toxins, breakdown products, mechanism of conferring antibiotic resistance, insertional inactivation
The main goal of cloing?
Produce recombinant molecules
Basic steps of cloning?
DNA isolation
Restriction enzymes digest to obtain gene of interest
Isolate fragments
ligation of vector and insert
Transformation-transformation of genetic information from one system to another by means of DNA.
Selection and identification
What are the requirements for vectors?
Must be relatively short molecule, msut be efficietnly replicated in host, must contain markers for selection, must contain a multiple cloning site where insert is placed, must have an origin of replication siet, utilized to direct replication machinery to recombinant molecule
Ligation reactions
Typically utilized a 3/1 ratio of insert to vector
Transformation
Into a host in replicate recombinant molecule. Utilizies electroporation, where an electrical current porates cells by opening ion gated channels
Cells for transformation need to be
grown in low salt, harvested in 10% glycerol to try to strip outer membrane of all protective charges
Transformation
Mix cells with DNA solution, place into a cuvette and shock them, 3-4 seconds; produces pores and DNA is taken up. Allow cells to recover in a very rich broth 37 C for one hour