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Lab Report #1 BICD 101

Lab Report #1 BICD 101

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Published by mwjackso
This is the first lab report of 3 in the BICD 101 class which was taken at UC San Diego for my BS in biochemistry and cell biology.
This is the first lab report of 3 in the BICD 101 class which was taken at UC San Diego for my BS in biochemistry and cell biology.

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Published by: mwjackso on Apr 04, 2009
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11/28/2012

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 Lab #1A02-92-6779- 1 -
Lab Report #1
Forward Genetics:
 An approach to the mapping and identificationof a mutation in trichome morphogenesis of 
 Arabidopsis thaliana
 Michael Wade JacksonBICD 101: Eukaryotic Genetics4/24/2003
 
 Lab #1A02-92-6779- 2 -
Introduction:
The desired outcome of this investigation was to apply a forward geneticsapproach to identify and map a possible genetic mutation in the trichome morphogenesisof 
 Arabidopsis thaliana
. The principle of a forward genetics approach is that one takes a phenotypic mutation present in the
 Arabidopsis
and then through the use of both acomplementation test and genetic mapping the gene or genes of interest in the sample population can be located. Forward genetics is: to start with a mutation and then identifythe responsible gene.The mutation of interest to this inquiry is the process of trichome morphogenesiswhich is first isolated in a process of mutant screening. The mutant of specific interest isthe two prong forming mutation which would play a role in trichome development and beinhibited in some fashion by some type of genetic mutation which would not be found inthe phenotypically normal plant. The trichome is an outgrowth of the epidermis such as ahair and in the
 Arabidopsis thaliana
the trichome is formed by one large cell. The two prong mutation once mapped to a specific site on one of the five chromosomes of 
 Arabidopsis
will provide insight into the developmental process behind trichomemorphogenesis.The benefits of understanding a developmental process such as this is both to aidein a greater understanding of the
 Arabidopsis
plant but as well the
 Arabidopsis
is itself amodel organism of genetic research and the mapping of its genome will allowcomparisons to be made amongst newly discovered research subjects and pre-existingmodel organisms.
 Arabidopsis
is used as a model organism due to its robust nature andease of growth in laboratory conditions as well as its cost effective nature. Theknowledge gained through the process of mutation identification and localization will provide a framework by which the developmental process of the trichome can be better understood and mutation that may arise may be targeted to prevent fatal mutations to the
 Arabidopsis
population.The process of mapping the mutation in Arabidopsis involved the use of both acoarse mapping and a fine mapping utilizing different genetic markers (polymorphisms).The coarse map was created by using eleven SSLPs (simple sequence length
 
 Lab #1A02-92-6779- 3 - polymorphisms) which utilize the pre-existing presence of tandem repeats of one- two- or three-nucleotide motifs. These are commonly called microsatellites and identificationcan be made by the variance in the number of repeats between two PCR primers. Thenumber and size of repeats is polymorphic between ecotypes which will allow the marker to be used as a co-dominant genetic marker. The benefit of SSLPs in the coarse geneticmapping is that they do not utilize restriction enzyme digests which add to the possiblelocations in which the experiment can go wrong.The fine mapping utilized CAPS (cleaved amplified polymorphic sequences)markers of which there were three. The CAPS markers utilize the presence of restrictionsites in sequences of DNA which is comparable to that of RFLPs (restriction fragmentlength polymorphisms). The benefit of using CAPS to RFLPs is that the CAPS can beassayed on agarose gels by electrophoresis and that the CAPS utilize PCR for hybridization instead of blot techniques. The fine map is developed once thechromosomal location of the mutation is known and linkage is shown to a SSLP marker and then CAPS surrounding the marker will be utilized based on sequence specificknowledge.The main diagnostic techniques used for both marker types are PCR and agarosegel electrophoresis which allow for amplification of a specific region of the genome andthen visualization of the size differences by the movement of DNA through agarose gel.The variance between the two markers is the need for restriction endonucleases in thecreation of diagnostic bands for CAPS markers.The trichome morphogenesis mutation of interest once mapped will allow awindow into the development of trichomes in
 Arabidopsis thaliana
which will provideinsight into future organisms of interest and elucidate the possible pitfalls anddevelopmental network of trichome development.
Materials and Methods:
 
The first step in mapping the mutation in trichome morphogenesis is the selectionof mutant phenotypes from a population of plants in a process known as a mutant screen.The mutant screen is created from irradiated seeds which produce mutations in singlecells within the developing plant creating a mosaic
 Arabidopsis
. The mosaic plant is then

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