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View the Research Proposal
View the completed Dissertation (NEW)
View some population simulation software (Diversion) (NEW)

See (right-click and save to download and view) the colloquium presentation prepared for La Sierra University (Having Fun With DNA.ppt), January 13, 2004 (NEW) - Quick overview of original project purpose and methods, followed by general steps involved in microsatellite molecular marker development.  Ends with explanation of Secondary Screening Method created and used.

*** This dissertation and all original data are available on a CD upon request. ***

Individuals Involved in the Study:

• Jeffrey A. Sonnentag (me)
• Ronald L. Carter
• Martin Wikelski
• William K. Hayes

• Sandra Orellana T.
• Jose Torres C.
• Franz X. Kümmeth
• Daniel G. Vergara G.
• Mónica X. Freire P.

• Determine what (if any) reproductive advantage females gain by mating with specific males
• Use of DNA fingerprinting to match males and females with offspring
• Evaluation of individual characteristics / survival
• Further characterize the mating organization and behavior of the marine iguana (Amblyrhynchus cristatus)

• Islet of Caamaño, Galápagos (Archipiélago de Colón), Ecuador

Where is that?!  (Click on the successive maps for greater location specificity)

• January-March, 1997
• Territorial males marked, measured, and blood sample taken (234)
• Daily observations of marked male territorial behavior
• Nesting females marked, measured, and blood sample taken (828)
• Additional observations of marked females and remaining marked males
• May-June, 1997
• Hatchling iguanas marked, measured, and blood samples taken (490)
• Additional observations of marked male and female behavior
• Recapture, repainting, and re-measuring of marked males found on the islet (39)
• Recapture and re-measurement of a sample of marked females (60)
• January-February, 1998
• Marking/remarking, measuring, and blood sampling (if necessary) of territorial males (30)
• Remarking of a sampling of marked females (50)
• Observations of marked male and female behavior
• Preliminary experiments . . . (concerning lek dynamics)
• Capture/recapture, measurement, and blood sampling (if necessary) of hatchlings (166)

• Created a genomic library (using Sau 3a restriction sites) of 300-700 bp DNA fragments
• Diagram of the basic process (extracting and cutting up the DNA, inserting DNA into plasmids/vectors, insertion of plasmids/vectors into bacteria for replication purposes, plating out and growth of bacterial colonies)  Another overview may be viewed by [clicking here]
• Selected colonies containing plasmids with inserted iguana DNA
• Overview of  blue/white colony selection technique
• Pictures of plates (petri dishes) containing blue and white bacterial colonies (Picture #1, Picture #2)
• Patching over/selecting white colonies for further growth and analysis
• Pictures of plates just started and after cultured (overnight @ 37^oC) bacterial growth
• Identification of bacterial colonies with plasmids containing inserted iguana DNA with specific repeated nucleotide sequences (microsatellites)
• Lifting/copying bacterial colonies onto a membrane
• Exposure of bacterial DNA (by breaking open/digesting the cells) and binding of DNA to the membrane
• Washing of membranes containing plasmid DNA with radioactive phosphorus (³²P) attached to a synthesized oligonucleotide that is complementary to (will bind to) a specific repeated DNA sequence
• Imaging of the membranes with the bound/attached DNA by exposing film to the radioactive ³²P that has been incorporated  (DNA with sites complementary to the synthesized repeated sequences binds the ³²P and appears as a dark spot on the developed film)
• Identification of bacterial colonies on the original plates that match up with the pictures
• Production of primers for microsatellite sequences
• Purification of plasmid DNA from colonies that have been identified as containing repeated sequences
• Diagram of the procedure used (from a purchased kit) to purify plasmid DNA
• Sequencing of iguana DNA in the plasmid
• Sample output from sequencing of a repeated mixed di- and trinucleotide (CA and CAC and/or CAA)
• Design of primers for PCR amplification of the regions of the DNA containing the microsatellite repeats to be used in paternity analysis (using a computer program [OLIGO] and sequences of the inserted iguana DNA)
• Testing of primers designed from DNA sequences
• Extraction of DNA from a subset of samples
• Test the synthesized (by GENOSYS) primers under numerous PCR conditions to determine whether they are useable (comparative pictures of results so far)
• Working on increased resolution of precise DNA fragment size
• Testing a 32 cm polyacrylamide gel
• Comparison of a 32 cm 8% polyacrylamide gel and a 16 cm 15% polyacrylamide gel
• Running the gel at room temperature vs. at 4^oC in the refrigerator
• Using copper plates to distribute heat evenly across the gel for equalized DNA movement over the gel's entire width
• . . . Bad buffer . . . OH NO!
• Increased gel resolution and decreased electrophoresis run time using Spreadex^® Polymer NAB
• Evaluated primers developed for detection of variation between individuals within the single population
• Submitted sequences identified so far to GenBank for use by others
• Discovered need for MORE microsatellite sequences so repeated the development process:
• Cut iguana DNA in small pieces
• Inserted iguana DNA into a circular plasmid (pUC19)
• Prepared compentent cells, that would accept foreign DNA
• Chemically transformed E. coli, putting the plasmid containing pieces of iguana DNA inside the bacteria
• Selected bacterial colonies with plasmids that had been inserted
• Screened selected bacterial colonies for potentially useful microsatellite repeat sequences
• Extracted plasmid DNA and performed secondary screening
• Sequenced promising looking iguana DNA inserts in plasmids
• Designed primers for sequences containing extended lengths of simple repeats
• Evaluated primers developed for utility in individual iguana identification (variablity in alleles between and within individuals)

• Dr. Ronald L. Carter - for financial, organizational, moral support
• Dr. Martin Wikelski - for provision of absolutely necessary organizational, contact, and communication needs
• Dr. William K. Hayes - for enthusiastic involvement and suggestions
• Dr. John F. Sands - for much needed laboratory directional assistance
• Charles Darwin Research Station - for necessary, and much appreciated, logistical support
• Parque Nacional Galápagos - for permits necessary for research activity and transport of blood samples
• INEFAN - for approval of necessary CITES permits for transport of blood samples
• Ecuadorian Consulate (of Los Angeles, California) - for providing visas when necessary
• TAME - for reduced flight rates for the research project's participants
• and most especially, the assistants who donated their time for field data collection (Sandra Orellana T., Jose Torres C., Franz X. Kümmeth, Daniel G. Vergara G., and Mónica X. Freire P.)

Jeffrey Sonnentag
Natural Sciences Department - Griggs Hall
Loma Linda University
Loma Linda, CA 92350
(909) 558-4530
(909) 799-5563 (home)
JSonnentag@yahoo.com

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