What allows effective and controlled expression of A and B insulin peptides?
cleaves insulin peptides at Met and A and B fragments are isolated
What holds the A and B chains of human insulin together?
Using the specificity of restriction endonucleases, selection of genes using specific antibiotic resistant genes
What allows the insertion of any gene into the plasmid for cloning?
Single stranded overhangs of cut DNA
Restriction digest, excising gene, inventory, annealing DNA fragment ends
describe the process of inserting a gene into a plasmid
Promoters, regulatory sequences, bacterial uptake
What do plasmids need in order to function?
How to bacteria take up plasmids?
Specialized DNA sequences in plasmid
What do mammalian cells require in order for the cloned gene to be expressed?
Must be transcribed
How are plasmids cloned in the nucleus of mammalian cells?
Antibiotic resistance and toxins
what is used to select for recombinant DNA?
Bacteria can make protein, or it can be put in mammalian cell for large scale protein production
how can a plasmid bring about protein production?
What is required for plasmids to be selected for?
Size of gene or its cDNA, modifications to protein by cell, delivery to the target tissue
What influences recombinant therapeutics?
Sequences that signal its secretion into the growth medium, be active so bacteria do not glycosylate proteins
What is required for an effective plasmid construct?
What is required to purify protein in manufacturing?
In culture supernatant
How is protein extracted from the growth medium in mammalian cells?
DNA fragments from bacterial, plant, or animal cells are fused to a vector and transferred to a host
Reverse transcriptase, alkali treatment (RNA breakdown), DNA polymerase
What are the steps in making cDNA?
transfer of DNA into mammalian cells
Amount of DNA for sequencing, transformation, restriction digestion, etc.
Amount of DNA for transfection, transformation, mutation, etc.
Spectrophotmetric analysis, restriction enzyme digestions, agarase gel electrophoresis
What methods are used to assess quantity and quality of DNA?
Subcloning or mutagenesis
How is DNA manipulated?
DNA is denatured (heated), primers attach to each strand and a new DNA strand is synthesized behind primers(5'->3') on each template strand and repeated
Describe the polymerase chain reaction
identification of DNA sequences by separating on gels and binding of specific complementary probe sequences by base-pairing
Radioactivity, fluorescence, or enzymatic reaction
What is used to detect hybridization in southern blotting?
monitors the fluorescence emitted during the reaction as an indicator of amplicon production at each PCR cycle
When does the reporter emit signal that is detected by a machine in Real-time PCR?