Chapter 5 - Exploring Genes and Genomes - part 1 Flashcards
Southern blots are used to separate and identify
DNA.
Northern blots are used to separate and identify
RNA.
de novo meaning
from the beginning.
Restriction enzymes
recognize specific base sequences in double helical DNA and cleave both strands of that duplex at specific places.
Restriction enzymes are used to
cleave DNA molecules into specific fragments that are more readily analyzed and manipulated than the parent molecule.
For most gels, the shorter the DNA fragment, the
farther the migration.
Western blotting refers to a technique for
detecting a particular protein by staining with specific antibody.
Stringency is
the required closeness of the match between primer and target.
Polymorphism
Instances of genetic variation.
RFLP stands for
restriction-fragment-length polymorphisms
Vector
A DNA molecule that can replicate autonomously in an appropriate host organism.
Vectors are designed to
enable the rapid, covalent insertion of DNA fragments of interest.
Plasmids
Naturally occurring circles of DNA that act as accessory chromosomes in bacteria.
A vector can be prepared for accepting a new DNA fragment by
cleaving it at a single specific site with a restriction enzyme
DNA ligase catalyzes
the formation of a phosphodiester bond at a break in a DNA chain
Cloning vectors are suitable for the
facile insertion and replication of a collection of DNA fragments
Cloning vectors often feature a polylinker region that includes many
unique restriction enzymes within its sequence
The polylinker region can be
cleaved with many different restriction enzymes providing great versatility in the DNA fragments that can be inserted
Reporter genes encode
rapidly-detectable markers such as antibiotic-resistance enzymes or fluorescent proteins
In blue-white screening, bacterial colonies will appear white when
DNA fragments are successfully inserted into the polylinker region
In addition to polylinkers, expression vectors often contain
sequences flanking the cloning site that simplify the addition of fusion tags to the protein of interest
In the lytic pathway of lambda phages, viral functions are
fully expressed, viral DNA and proteins are quickly produced and packaged into viral particles leading to the destruction of the host cell
In the lysogenic pathway of lambda phages, the phage DNA becomes
inserted into the host-cell genome and can be replicated together with the host cell DNA for many generations, remaining inactive
Lambda particles can only take up DNA measuring from _____ to _____ of the normal genome
78-105%
To clone a gene that is present just once in an entire genome, two components must be available
- A specific oligonucleotide probe for the gene of interest
- A DNA library that can be screened rapidly
For the human genome, a 99% probability of success using the genomic library requires screening about _______ _____ clones
500,000
The key to forming complementary DNA is
the enzyme reverse transcriptase
Complementary DNA molecules can be inserted into expression vectors to
enable the production of the corresponding protein of interest
In expression cloning, clones of cDNA can be screened on the basis of
their capacity to direct the synthesis of a foreign protein in bacteria
New genes can be constructed with designed properties by making what three kinds of directed changes?
- deletions
- insertions
- substitutions
Mutant proteins with single amino acid substitutions can be readily produced by
site-directed mutagenesis
In cassette mutagenesis,
a variety of mutations including insertions, deletions, and multiple point mutations can be introduced into the gene of interest
Inverse PCR is used to introduce
deletions into plasmid DNA
Novel proteins can be created by splicing together
segments that encode domains that are not associated in nature
