GOALS OF THE LABORATORY:
The goal of this laboratory is to provide participants with: (1) an understanding of
the molecular and cellular basis of developmental processes and mechanisms; (2) a
solid working knowledge of the basic molecular techniques and procedures commonly
used in molecular biology research labs; (3) a firm foundation in the theoretical aspects
of molecular biology which make all these techniques “work”; (4) a sense of how to
design and carry out a research project. Finally, last but not least, an important aim of
this laboratory is to produce interesting new data on potentially novel developmental
genes. This will be accomplished by a semester-long project project involving the
identification, cloning, and characterization of potentially developmentally-significant
genes, that is, genes which play a critical role in some aspect of normal vertebrate
embryogenesis. More specifically, this laboratory will focus on cloning genes involved
in neuronal signaling, genes that have been implicated in developmental decisions.
We will clone these genes from Xenopus laevis, an amphibian model system widely
used in developmental biology research, and Peromyscus, a rodent model system
widely used in ecological and physiological investigations.
The overall strategy will involve cloning these genes using PCR (polymerase
chain reaction) screening as our primary approach, but also employing homology
screening of a genomic library.
The polymerase chain reaction allows the investigator to amplify, isolate, and
clone specific pieces of DNA from a small amount of target DNA. This is accomplished
by designing and synthesizing DNA primers that flank the region of interest and
essentially serve as the priming fragment for DNA replication in vitro. Following
amplification, the PCR product can be cloned and subsequently characterized to
determine its sequence. This procedure involves the following steps.
Primer Design: discussion and analysis
of how the investigator designs primers for a
specific gene or fragment of a gene; this is done by analysis of existing sequence
information from Genbank or from the primary literature.
DNA Extraction: extraction and purification
of genomic DNA from Xenopus for use in
Genomic DNA Analysis: test the quality
and quantity of the genomic DNA by restriction
digest and gel electrophoresis.
Polymerase Chain Reaction: set up PCR using
designed primers to amplify gene
fragment of interest.
PCR Product Analysis: analyze products obtained from PCR via gel electrophoresis.
Cloning of PCR Fragment: PCR products
will be cloned into a plasmid vector using the
“TA Cloning Kit” (Invitrogen); this involves a ligation into the vector and transformation
into bacterial cells.
Mini-Prep Analysis: bacterial colonies
(containing plasmid with insert) will be cultured
and “mini-preps” performed to isolate plasmid DNA; the plasmid DNA will be digested
with appropriate enzymes to determine the presence and size of the insert.
Sequence Analysis: using dideoxy sequencing
(Sequenase, USB) and NCBI
databases, your PCR products will be sequenced and analyzed
GENOMIC LIBRARY SCREENING:
Library screening is an alternative, though not necessarily exclusive, way to
isolate a gene. Frequently PCR products are used in library screening to obtain a
longer (or complete) version of the gene. There are two types of libraries: (1) cDNA
libraries, which are reverse transcribed from mRNA and therefore contain only
transcribed sequences, and (2) genomic libraries which theoretically include the entire
genome of the organism. Given that we do not know exactly when and where our
genes are expressed, we will be screening a genomic library. There are two basic
approaches to library screening. Differential screening is used if the investigator does
not know in advance what type of gene is being sought or if a closely related gene does
not exist. For example, if one is interested in identifying genes which are involved in the
development of the brain, a brain library could be differentially screened with probes
from brain and, for example, kidney. Those sequences which only hybridize to the
brain probe are likely to yield novel brain-specific genes. Homology screening involves
using a probe from a gene which is presumably homologous to the one you wish to
clone – this is performed at “low stringency” to isolate similar or related genes. This
approach is useful if the investigator knows in advance what type of gene is being
sought and if a closely related gene is already available. The basic steps of this
procedure are outlined below:
Plating the Library: the library will be
plated at various dilutions on agar plates to attain
an appropriate density of plaques.
Membrane “Lifts”: nylon membranes
will placed atop the plates to transfer the plaques
to the membrane. Membranes will then be placed into a pre-hybridization solution.
Probe Synthesis: DNA for the probes must
be isolated by cutting it our from the plasmid
with restriction enzymes, running the digest on a gel, cutting out the appropriate
fragment, and “gene-cleaning” it. This DNA is then used for radioactive probe
Hybridization: the membrane is then”hybridized”
by incubating the membrane in a
solution containing the probe. The filters are then washed and placed on X-Ray film.
Film Development and “Positive Plaque Identification”:
films are developed and
“positive” plaques are removed.
Plaque Purification: probing is repeated until a single pure plaque is isolated.
Characterization of Positive Plaques: a
phage prep is performed to isolate phage DNA
and the DNA then subjected to Southern blot analysis and subsequent subcloning into
plasmid vectors for sequence analysis.
Our official meeting times are Tuesday or Wednesday afternoons, but we will
frequently need to perform various procedures during other hours because of
experimental constraints. These hours will be flexible. Grading will be based upon
attendance, enthusiasm, attention to experimental detail (1/4), your lab notebook (1/4),
a final lab report (1/4), and a brief, oral lab final (1/4). Given the goal of producing
usable scientific results, you will be encouraged to do as much as you like with your