Lecture 17 - Recombinant DNA technology Flashcards
Recombinant DNA (rDNA) molecules are
DNA molecules formed by laboratory methods of genetic recombination
In cloning, fragment and plasmid DNA is treated with
EcoRI and NotI restriction enzymes to create sticky ends
Two DNA molecules, cleaved with a common restriction enzyme such as EcoRI, can be ligated to form
recombinant molecules
If a DNA fragment doesn’t contain a site for a restriction enzyme,
a short, chemically synthesized DNA linker with a restriction enzyme cleavage site can be added
A vector is a
DNA molecule, which is used as a vehicle to carry foreign genetic material into a cell, where it is replicated and/or expressed
In general, there are 2 types of DNA vector
- Cloning vectors
- Expression vectors
Cloning vectors are used to make
numerous copies of the inserted foreign DNA
Expression vectors are used to
express large amounts of protein from the inserted foreign DNA
Cloning vector examples (2)
- Plasmids
- Bacteriophage
Plasmids are
naturally occurring extrachromosomal DNA
A cloning vector contains what 3 things?
- Origin of replication
- Selection marker
- Restriction sites
Promotor sequences are designed to
drive the transcription of large amounts of a protein-coding gene
The gene of interest in the expression vector often contains
fusion tags to easily purify the expressed protein
Expression vectors often contain a polylinker region that includes
several restriction sites
Nuclease cleavage at a restriction site linearizes the circular plasmid so that
a foreign DNA fragment can be inserted
Recombinant plasmids are hybrid DNA molecules consisting of
plasmid DNA sequences plus inserted DNA elements (such hybrid molecules are called chimeric plasmids)
The two primary uses for alkaline phosphatase (AP) in DNA manipulations:
- Removing 5’ phosphates from plasmid and bacteriophage vectors that have been cut with a restriction enzyme
- In subsequent ligation reactions, this prevents self-ligation of the vector
Alkaline phosphatase removes
5’ phosphate groups from DNA and RNA
APs are most active at _____ pH
alkaline
Sources of alkaline phosphatase (3)
- Bacterial alkaline phosphatase (BAP)
- Calf intestinal phosphatase (CIAP)
- Shrimp alkaline phosphatase
Often, one desires to insert foreign DNA in a particular
orientation
Transformation in bacteria is the
uptake and incorporation of exogenous genetic material through the cell membrane
Transformation requires
competent cells
Competent cells are in a state in which
DNA molecules can pass through the membrane
Viral vectors are used to
get a vector into a bacterial cell
In viral vectors, a ________ is often used
lambda phage
Transfection
In mammalian cells, the process of uptake and incorporation of exogenous genetic material
3 ways DNA gets through the membrane in transfection
- Physical transfection
- Chemical transfection (most common)
- Biological transfection
Researchers use Ti-plasmid derivatives to
deliver foreign genes into plant cells
Site-directed mutagenesis is a technique for
studying protein structure-function relationships, gene expression, and vector modification
In site-directed mutagenesis,
the sequence of a cloned DNA molecule can be altered to produce specific changes in the expressed gene product
Oligonucleotide-directed mutagenesis is commonly used to
produce specific single-base changes in a cloned gene
Oligonucleotide mutagenesis employs a
short primer sequence that is complementary to the gene except for one mismatched base
