PCHS students preparing for gel electrophoresis.
Polk County High School lies in the shadow of picturesque mountains in Western North Carolina. Polk County occupies a unique thermal belt and exhibits a broad range of interesting flora and fauna. The school is rural and the student population is typical for North Carolina.
What is not typical for Western North Carolina is an out-population of Sweetbay Magnolia (M. virginiana). This species normally is indigenous to coastal areas but an out-population exists in Polk County. Due to the unique location of this population, PCHS has embarked in a research project entitled the Magnolia Detectives Project. Project leader, Jennifer Allsbrook , PCHS Science Department Chair and biology instructor, and Mr. John Vining of the North Carolina Cooperative Extension Service, have developed the project to determine the genetic relationship of this out- population to others stands of Sweetbay Magnolia in North and South Carolina.
In the photo to the left, PCHS students are collecting leaves from the Polk County stand of Sweetbay Magnolia as community partner, John Vining, coordinates the effort. The tree in the foreground is a specimen of Magnolia virginiana.
Funding for this project has been provided by generous grants from the following grant-making institutions/programs:
$2000 - ING Unsung Heroes Grant Award
$15,000 - Toshiba American Foundation - Science & Math Education Grant
Aaron Green and Jennifer Allsbrook (far right) accept $15,000 from Lisa Allen of Toshiba.
$2000 - North Carolina Biotechnology Center - Educational Enhancement Grant
Project Objective: To determine the closest genetic relatives of the Polk County stand of Magnolia virginiana.
Strategic Overview: Students in the Introduction to Biotechnology class will be extracting DNA from leaves collected from Magnolia virginiana in Polk County and from neighboring locations in North and South Carolina. This DNA will then be amplified through PCR (polymerase chain reaction) using an ISSR marker system. An SSR is a short or simple sequence repeat that usually involves dinucleotide or trinucleotide tandem repeats. For example, the sequence CTCTCTCTCTCTAG may be found many times within the genome of an organism. These regions of repetitive DNA can be used to amplify regions of the genome using PCR, but this requires significant knowledge of the nucleotide sequences flanking them within the genome. To get around this problem, the ISSR technique can be utilized. This ISSR (inter-simple sequence repeat) technique utilizes a single primer that will anneal to the SSRs at two locations in the genome and then the region between these SSRs is amplified. Since the locations of the SSRs are highly variable or polymorphic, this approach can be used to create DNA fingerprints of individuals and can be used to do genomic comparisons within and among populations. It is through this approach the the PCHS Magnolia Detectives hope to determine the closest relatives to the Polk County out-population of Sweetbay Magnolia.
The Introduction to Biotechnology class worked diligently to learn the techniques and laboratory protocols necessary to be successful with the Magnolia Detectives Project. We started the semester learning about biotechnology basics, DNA structure and function, and DNA manipulation techniques. Some of the laboratory experiments that we conducted included:
1) DNA necklaces from cheek cell DNA extraction
2) Restriction enzyme digestion and gel electrophoresis RFLP (restriction fragment length polymorphism) analysis
3) PCR (polymerase chain reaction)
4) Southern blotting and nucleic acid hybridization
5) DNA fingerprinting using VNTR (variable number tandem repeat), Alu, and mDNA markers
6) Bacteria Transformation with GFP (green florescent protein) and BFP (blue florescent protein)
7) GFP and BFP protein purification using gel filtration chromatography and size determination with gel electrophoresis
Follow our progress and share our experiences at the PCHS Magnolia Detectives blog!