chapter 15 Flashcards
What is recombinant DNA technology?
A set of techniques used to amplify, maintain, and manipulate DNA sequences in vitro and in vivo.
List applications of recombinant DNA
- Fragment and purify DNA
- Replicate DNA fragments in vitro
- Combine fragments into recombinant molecules
- Sequence DNA
- Identify complementary sequences
- Introduce DNA into organisms
- Assay effects of introduced DNA” cell
What are restriction enzymes?
Nucleases that recognize specific DNA sequences and cut both strands at that site, often producing sticky ends
What are sticky ends in restriction digestion?
Single-stranded overhangs created by certain restriction enzymes that allow complementary base pairing between fragments
How do you estimate restriction enzyme cut frequency?
Use the formula 4^n where n = number of bases in the recognition sequence.
What is restriction mapping
Analyzing DNA fragment sizes after restriction digestion using gel electrophoresis to map enzyme cut sites
What is molecular cloning?
The process of creating and inserting recombinant DNA into a vector to be amplified in a biological system
Steps in molecular cloning
1.Ligate DNA fragment and vector with DNA ligase
2. Transform plasmid into competent cells
3. Allow replication in host
What is transformation in cloning?
Introducing recombinant plasmid into competent E. coli cells using chemical treatment and heat shock
How do you select transformants?
Plate cells on antibiotic-containing media; only those with the plasmid (e.g., with ampicillin resistance) will grow
How do you confirm insertion of the DNA fragment?
Use blue-white screening with X-gal: white colonies contain the insert, blue colonies do not.
How else can you verify the insert?
Digest plasmid DNA and use gel electrophoresis to check for the expected fragment size
Why can’t all genes be expressed in E. coli?
E. coli lacks machinery for post-translational modifications needed by some eukaryotic proteins
What is an eukaryotic expression system?
Vectors designed for use in eukaryotic cells like yeast or tissue culture, with appropriate regulatory elements
How was human insulin produced in E. coli
Synthetic DNA for insulin A and B chains was expressed in E. coli using plasmids fused to the lacZ gene
What is the two-chain method of insulin expression?
Separate plasmids express insulin A and B chains as fusion proteins with lacZ. Cyanogen bromide cleaves the insulin chains
What is a fusion protein?
A protein made from a fusion gene, combining segments from different genes
What is cyanogen bromide used for?
To cleave fusion proteins at methionine residues to isolate the insulin chains
What induces transcription in insulin expression
Lactose in the absence of glucose induces transcription of the fusion genes
How are transgenic animals generated
Injecting vectors or mRNA/proteins into embryos; the transgene must integrate into germline cells to be inherited
What was Dolly the Sheep and how was she cloned?
The first cloned animal; her nucleus from a somatic cell was inserted into an enucleated egg and electrically stimulated
Is Dolly genetically identical to the nucleus donor?
No. She shares nuclear DNA but has mitochondrial DNA from the egg donor; epigenetic factors may also diffe
