Remainder of Chapter 9 + 10 material
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What is a plasmid?
Plasmids: a plasmid is a circular DNA molecule that replicates separately from the host chromosome. They are tools used for the transfer.
What is transfection?
Transfection: this is when you introduce plasmids into cells. The ability to get vectors into cells.
Define cloning vector:
Cloning vector: used for making many plasmid copies. Also thought of as a self-replicating molecule.
What is a construct?
Construct: This is what a recombined plasmid is called.
Transformation: Introduction of an exogenous DNA into a cell, causing the cell to acquire a new phenotype. Process in which small plasmids can be introduced into bacterial cells.
How is a vector delivered into cells?
How a vector is delivered into cells: cells take up plasmid by heat shock or a high voltage pulse.
Describe recombinant DNA technology:
Recombinant DNA technology: cloning entails cutting DNA into fragments with enzymes; selecting and possibly modifying a fragment of interest; inserting the DNA fragment into a suitable cloning vector; transferring the vector with the DNA insert into a host cell for replication; and identifying and selecting cells that contain the DNA fragment.
Blunt ends: caused by restriction endonucleases cleavingboth strands of DNA at opposing phosphodiester bonds, leaving no unpaired bases on the ends.
Sticky ends: why are they preferred?
- Sticky ends: Two DNA ends in the same DNA molecule, or in different molecules, with short overhanging single-stranded segments that are complementary to one another, facilitating ligation of the ends.
- Sticky ends are preferred for cloning, because the base paring of complementary sticky ends greatly facilitates the ligation reaction.
Expression vector: cloning vectors with the transcription and translation signals needed for the regulated expression of a cloned gene.
Cloning vector: a small carrier molecule of DNA capable of self-replication.
How do cloning and expression vectors differ?
When you use an expression vector, you produce the protein of the gene that is to be cloned, where in a cloning vector you just clone the gene.
What are the structural properties of nucleotides?
Structural properties of nucleotides: Backbones are hydrophilic (pentose hydroxyls form hydrogen bonds with water), phosphates are completely ionized at pH 7.0 = negative charged, and bases "stack" to minimize contact with water.
Chemical properties of nucleotides that explain DNA structure:
Chemical properties of nucleotides that explain DNA structures: (1) bases are hydrophobic (2) H-bonds form between nucleotide bases of two different strands (3) Both 1 and 2 stabilize binding between two or more strands. A + T make two hydrogen bonds and C + G make 3 hydrogen bonds.
Know how hydrogen bonds hold DNA strands together in duplex, triplex, and quadriplex forms:
Duplex DNA: H-bonds form between nucleotide bases of two different strands.
- Triplex DNA: b-form of DNA makes additional H-bonds in major groove. Special hydrogen bonds: Hoogsteen positions (the atoms that make the bonds). Non-Watson-Crick pairing: Hoogsteen paring (the bases that make the bonds). The Hoogsteen pairings add stability to DNA and the 3rd strand lays in
- major groove.
- Tetraplex DNA: 4-stranded DNA where there is a high
- proportion of Guanosine residues, it is very stable. It occurs at end of
- chromosomes to stabilize DNA. Just think 4G.
Hybridization: ability to make and break H-bonds using complementation
What is a eicosanoid? what are its classes?
- Eicosanoid: intercellular messengers.
- Has 3 defined classes: prostaglandins, thromboxanes, and leukotrienes.
Prostaglandins: have one 5-carbon ring.
Thromboxanes: one six-carbon ring.
Leukotrienes: contain 3 conjugated double bonds, open (no cyclic) structure.
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