Running head: Molecular Biology – BIO3001 1
Molecular Biology
BIO3001
Summary
Name
Institution
Course
Tutor
Date
, Molecular Biology – BIO3001 2
BIO3001 – Molecular Biology
Table of Contents
, Molecular Biology – BIO3001 3
Animal Models & Molecular Techniques
Gene analysis: Connecting knowledge about sequence, structure and
biochemical activity of encoded protein to its function in the context
of a living cell or organism is important. This is done by examining
phenotypic consequences of mutations that inactivate a particular
gene.
Classical genetics: isolation of a mutant that appears to be
defective in the process of interest. Genetic methods are then
used to identify and isolate the affected gene. The isolated gene
can be manipulated to produce large quantities of the protein it
encodes for biochemical experiments and to design probes for
studies of where and when the protein is expressed in an
organism.
Reverse genetics: identification and isolation of a gene of an
interesting protein, which is then altered and reinserted into an
organism to study it effects.
Mutations can have different effects:
Recessive mutant: often causes a loss of function.
Dominant mutant: often causes a gain of function. Can be
identified by mating two homozygous strains (75% of F2 has
mutant phenotype)
Dominant negative mutation: causes production of a protein
that inactivated the products of the wild-type gene. Produces
phenotype similar to loss of function mutation.
Cell isolation
, Molecular Biology – BIO3001 4
Fluorescence-activated cell sorting (FACS) is a specialized
type of flow cytometry which provides a method for sorting a
heterogeneous mixture of biological cells into two or more
containers, one cell at a time, based upon the specific light
scattering and fluorescent characteristics of each cell. The
wanted cells are conjugated with antibody-associated
fluorescent dye (fluorophore) and when a laser is pointed at the
mixture, light gets excited at a certain wavelength. Cells with an
excitation above a certain wavelength (wanted cells) are
separated.
Different molecular genetic techniques can be used to isolate,
sequence and manipulate genes and to analyze when and where in the
cell this protein is expressed.
Identification: done by genetic screening
Conditional mutations: used to study phenotypic expression of
a mutation in essential genes (usually lethal mutations, tricky to
study)
o Temperature sensitive mutations: useful in bacteria and
lower eukaryotes (haploid yeast) that can grow at a range
of temperatures. The mutant phenotype is observed at the
nonpermissive temperature and not observed at the
permissive temperature (usually higher), even though the
mutant allele is present. This temperature-sensitive mutant
produces an altered protein that works at the permissive
temperature but unfolds and is nonfunctional at the
nonpermissive temperature. Mutant strains can thus be
maintained at permissive temperature, after transferring
Molecular Biology
BIO3001
Summary
Name
Institution
Course
Tutor
Date
, Molecular Biology – BIO3001 2
BIO3001 – Molecular Biology
Table of Contents
, Molecular Biology – BIO3001 3
Animal Models & Molecular Techniques
Gene analysis: Connecting knowledge about sequence, structure and
biochemical activity of encoded protein to its function in the context
of a living cell or organism is important. This is done by examining
phenotypic consequences of mutations that inactivate a particular
gene.
Classical genetics: isolation of a mutant that appears to be
defective in the process of interest. Genetic methods are then
used to identify and isolate the affected gene. The isolated gene
can be manipulated to produce large quantities of the protein it
encodes for biochemical experiments and to design probes for
studies of where and when the protein is expressed in an
organism.
Reverse genetics: identification and isolation of a gene of an
interesting protein, which is then altered and reinserted into an
organism to study it effects.
Mutations can have different effects:
Recessive mutant: often causes a loss of function.
Dominant mutant: often causes a gain of function. Can be
identified by mating two homozygous strains (75% of F2 has
mutant phenotype)
Dominant negative mutation: causes production of a protein
that inactivated the products of the wild-type gene. Produces
phenotype similar to loss of function mutation.
Cell isolation
, Molecular Biology – BIO3001 4
Fluorescence-activated cell sorting (FACS) is a specialized
type of flow cytometry which provides a method for sorting a
heterogeneous mixture of biological cells into two or more
containers, one cell at a time, based upon the specific light
scattering and fluorescent characteristics of each cell. The
wanted cells are conjugated with antibody-associated
fluorescent dye (fluorophore) and when a laser is pointed at the
mixture, light gets excited at a certain wavelength. Cells with an
excitation above a certain wavelength (wanted cells) are
separated.
Different molecular genetic techniques can be used to isolate,
sequence and manipulate genes and to analyze when and where in the
cell this protein is expressed.
Identification: done by genetic screening
Conditional mutations: used to study phenotypic expression of
a mutation in essential genes (usually lethal mutations, tricky to
study)
o Temperature sensitive mutations: useful in bacteria and
lower eukaryotes (haploid yeast) that can grow at a range
of temperatures. The mutant phenotype is observed at the
nonpermissive temperature and not observed at the
permissive temperature (usually higher), even though the
mutant allele is present. This temperature-sensitive mutant
produces an altered protein that works at the permissive
temperature but unfolds and is nonfunctional at the
nonpermissive temperature. Mutant strains can thus be
maintained at permissive temperature, after transferring
