Download Purification/UV-Vis Analysis Polymerase Chain Reaction (PCR

DETERMINING BREEDING PATTERNS OF Odocoileus
virginianus (white tailed deer) BY NUCLEAR DNA
FINGERPRINTING.
Richard J. Toth*, Michelle A. Peck*, Durwood B. Ray PhD., Fredrick J. Brenner PhD.
Grove City College, 100 Campus Drive, Grove City, PA 16127
Department of Biology
Primer Sets
Nuclear DNA
Abstract
We intend to study nuclear DNA from wild herds of white-tailed deer (Odocoileus
virginianus) as a means to track maternal and paternal breeding history within various
populations located in Ohio and Pennsylvania. Thirty-three samples, representing a large
variety in terms of age and sex, were procured via parks. DNA was originally obtained
from liver tissue, but the experimental procedures can utilize other sources such as hair or
blood samples. Polymerase Chain Reaction (PCR) was used in conjunction with our own
unique batch of fluorescently labeled primers in order to amplify 10 different short tandem
repeat (STR) segments of the deer DNA. Eight nonfluorescent primer sets are currently
being studied in order to increase our deer variability predictions. Most of our primer sets
have shown to work in closely related species. Gel electrophoresis was utilized to select
desired PCR products. An Applied Biosystems 310 single capillary automatic DNA
sequencer was used to analyze the nuclear STR regions to create a unique DNA profile
for individual deer, which is analogous to forensic identification of human nuclear DNA.
Based on allelic size and location, the DNA profiles are currently being used to identify
individual members within a complete herd of wild deer. One future objective is the
combination of threeSTR regions into a single reaction mixture for a more rapid and
efficient GeneScan analysis. The ultimate goal remains to study the breeding patterns of
localized white-tailed deer herds for genetic variability and to determine the major
fraternal contributor of each herd.
Methods
•Extract and isolate nuclear DNA from source
•Run individual PCR reactions using fluorescently-tagged primer sequences to amplify
the desired short tandem repeats (STRs) sequences
•Analyze by gel electrophoresis in a 2% Agarose E-gel (Invitrogen)
•Quantify the PCR products using NanoDrop® Photospectrometer
•Load in sequencer
•Analyze results from the electropherogram
Nuclear DNA extraction
•Take 0.5g of tissue liver sample suspended in 20mL of NET(0.1M NaCl in 1mM
EDTA[standard]), 20mM Tris-HCL, pH 7.4 solution
•Add 2mL 20% SDS into same 250mL Erl. Flask and incubate with occasional swirling in
60°C water bath for 10min.
•Cool to room temperature and add 5mL 6M NaClO4. Stir the mix gently
•Transfer mix to 250mL polyethylene centrifuge and add an equal volume of ChCl3
•Gently mix and invert for 5min and then centrifuge for 10min @3000-5000 rpm
•Carefully remove the upper aqueous layer. Repeat this step again
•Spool out DNA using a glass rod and put the nuclear DNA into a TE solution
•Gently layer 37.5mL of ice-cold 95%ethanol by decantation over a glass rod to the inside
wall of the beaker
•Immerse tip of the glass rod to interface region between alcohol and aqueous solution.
Stir clockwise to locally mix the layers. (alternative method=use centrifuge)
•Put DNA in freezer at -20°C
Primers:
OvA
OhD
OvH
OhN
OhP
Rt05
Rt07
Rt13
Rt24
Rt30
BL42
BM4107A
BM6506
OvirA
BM1225
BM4208
OheQ
Sika amelogenin
Species of origin:
O. virginianus (white-tailed deer)
O. hemionus (mule deer)
O. virginianus (white-tailed deer)
O. hemionus (mule deer)
O. hemionus (mule deer)
Rangifer tarandus (caribou)
Rangifer tarandus (caribou)
Rangifer tarandus (caribou)
Rangifer tarandus (caribou)
O. hemionus (mule deer)
Bos taurus (domestic cow)
Bos Taurus (domestic cow)
Bos Taurus (domestic cow)
O. hemionus (mule deer)
O. hemionus (mule deer)
O. hemionus (mule deer)
O. hemionus (mule deer)
O. hemionus (mule deer)
Di-deoxy dye-terminator reaction
•The di-deoxy dye-terminator reaction is run much like a PCR, except instead of dNTPs ddNTPs are used. A ddNTP is a
nucleotide without an OH at the 3’ Carbon of the sugar ring. In addition thse ddNTPs are labeled with a color, different for
each ddNTP.
•This color is the color the sequencer picks up, during the electrophoresis in the 310 DNA sequencer
•Since there is a lock of a 3’ OH, there can be no addition of nucleotides to a ddNTP. Therefore, by probability, in a segment
of DNA approximately 700bp long, every possible combination of termination will be made.
DNA
Sequencing
Gel Electrophoresis
ABI Prism 310 single capillary automatic DNA sequencer
•On a sheet of parafilm, create a mixture
for each PCR trial that consists of
•9µL of triple distilled water
•5µL Promega blue/orange dye
•6µL PCR sample
•Add each sample to the individual wells
on the E-gel
•Place 20µL of Trackit standard ladder into
the last well on the E-gel
•Run in a 2% Agarose E-gel (invitrogen)
containing ethidium bromide for 30min
•Remove the gel and take a UV picture of
the samples in Bio-Rad imaging unit
Results
®
NanoDrop
•In our studies, we found that the sizes of the PCR products produced by the collection of nuclear
DNA primer pairs in one white-tailed deer (#126444) to range from 163 to 300 base pairs.
Photospectroscopy
•UV-vis is a quick and accurate way of determining the
purity/concentration of the DNA, it is able to measure spectra from a 1.0
microliter sample in 10 seconds
•In purity assessment, two peaks are being evaluated at 260nm and
280nm
•At 260 nm DNA absorbs due to the electron density in nucleotide bases
•At 280 nm proteins absorb due to the electron density in the side chains
of Phe, Trp, Tyr
•A ratio of the intensities will give 260/280 = 1.8 if perfectly pure
NanoDrop® ND1000 Photospectrometer
•Successful GeneScan on primers: OhN, OhD, OhP, BL42, Ova, Rt13, Tr24, Rt05, Rt07, and OvH.
Conclusions
•We have completed Gene Scans on 10 of the 18 primer sets at this time. The ones we have tried have shown
good results.
•Future analysis of the remaining 8 primers, including the amelogenin primer for sex determination, will help further
differentiate the individual deer.
•The 18 different STRs of the nuclear DNA will be able to correctly identify each individual deer as a unique member
of the herd by noting allelic variations.
Future Work
•Verify primers BM6506, BM4107
•Analyze primers OvirA, Rt30, BM1225, BM4208, OheQ, and sika amelogenin (sex-determining) for STR loci
•Extract DNA from other white tailed deer liver samples to create a greater sample size
•Develop a single procedure to create an individual profile for each member of the herd
•Use these STR lengths to produce a unique identification profile for the white-tailed deer (similar to human DNA
identification)
Polymerase Chain Reaction (PCR)
•The PCR reaction runs: 95°C for 10 mins.; 40 cycles of 94°C for 1 min., 57°C for 1.0 min.,
and 72°C for 1 min.; 72°C for 6 mins.; 4°C
•The following are combined and placed in a thermocycler:
○nuclear DNA
○PCR Buffer with Mg
○dNTPs in large excess
○Taq Gold: A thermophillic DNA polymerase that is able to survive high temperatures
during denaturation
○Primers specific to desired regions of the nuclear DNA
Purification/UV-Vis Analysis
•Purification of PCR primers is accomplished by a QIAquick kit protocol
(another version of column chromatography)
•The purification is determined by UV-Vis analysis
•Evaluate the primer sets to identify allelic size profiles of individual members of a complete herd of wild deer
obtained in Ohio so that we can determine the major fraternal contributor to each herd
Acknowledgments
•Research was funded by the Grove City College Swezey Fund and enabled by use of GCC Rockwell laboratories.
•Special thanks to Laurie Lytle, Lab Technician.
•Special thanks to Megan Osborne and Emily Vroegindewey for their preliminary work in this project.