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Lecture 6
Some More Techniques in Biotechnology
Issues in Biotechnology:The Way We Work With Life
Dr. Albert P. Kausch
life edu.us
The Mechanics of DNA
Issues in Biotechnology:Biotechnology, Our Society and Our Future
OnCampus LiveOnCampus LiveBCH 190, MIC 190, AFS 190, NRS 190, PLS 190BCH 190, MIC 190, AFS 190, NRS 190, PLS 190
OnLine BCH 190OnLine BCH 190
A Sweeping General Survey on Life and BiotechnologyA Public Access College Course
The University of Rhode Island
Kimberly Nelson
Issues in Biotechnology:The Way We Work With Life
Dr. Albert P. Kausch
life edu.us
© life_edu
Section I. The Mechanics of Life and General Biotechnology
Section II. The Applications of Biotechnology
A Sweeping General Survey on Life and BiotechnologyA Public Access College Course
The University of Rhode Island
BCH 190BCH 190
life edu.us
Kimberly Nelson
Issues in Biotechnology:The Way We Work With Life
Dr. Albert P. Kausch
© life_edu
5. Trends, Patterns and Relationships in Biology
6. Some More Techniques in Biotechnology
A Sweeping General Survey on Life and Biotechnology
A Public Access College Course
The University of Rhode Island
Issues in Biotechnology:The Way We Work With Life
Dr. Albert P. Kausch
life edu.us
The Mechanics of DNA
© life_edu
Lecture 6
Some More Techniques in Biotechnology
Issues in Biotechnology:The Way We Work With Life
Dr. Albert P. Kausch
life edu.us
The Mechanics of DNA
© life_edu
Are there any foods or ingredients that you have avoided or eaten less of?
(A) yes(B) no(C) do know(D) refuse to answer
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Half of consumers are avoiding some Half of consumers are avoiding some food/ingredient…food/ingredient…
Are there any foods or ingredients that you have avoided or eaten less of?
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If yes, what foods or ingredients did you avoid or eat less of?
Foods/ingredients avoided
(A) sugar / carbohydrates(B) fats / cholesterol(C) animal products(D) salt / spices(E) biotechnology products
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……it’s not biotech! it’s not biotech! If yes, what foods or ingredients did you avoid or eat less of?
(Open-ended; Multiple responses allowed, n = 478)
IFIC 2005
Foods/ingredients avoided 3/05
Sugar/Carbohydrates 58%
Fats/Cholesterol 37%
Animal Products 34%
Salt/Spices 14%
Snack Foods 11%
Biotechnology < ½ %
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FDA requires special labeling when a food is produced under certain conditions: when biotechnology’s use introduces an allergen or when it substantially changes the food’s nutritional content... Otherwise special labeling is not required. Would you say that you support or oppose this policy of FDA?
(A) support(B) oppose(C) neither support or oppose(D) don’t know(E) refuse to answer
Current FDA labeling policyCurrent FDA labeling policy supported by majority supported by majority
0%
10%
20%
30%
40%
50%
60%
70%
80%
Don'tknow/refused
Neither supportnor oppose
Total oppose
Total support
FDA requires special labeling when a food is produced under certain conditions: when biotechnology’s use introduces an allergen or when it substantially changes the food’s
nutritional content... Otherwise special labeling is not required. Would you say that you support or oppose this policy of FDA?
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It is now possible to clone any gene from any
organism and move it into plants
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Plasmids are circular pieces of DNA found in some bacteria
Many copies per cell
Antibiotic resistance gene
Plasmids can be cut and pasted back together
Foreign genes can be inserted
Enzymes were discovered that cut DNA at specific sequences
And subsequently, enzymes were discovered that paste DNA together
The ability to cut and paste DNAallowed gene cloning
How is a gene cloned?How is a gene cloned?Boyer, Cohen, and Berg, 1972
How is a gene cloned?Foreign DNA (gene)is inserted into a plasmid that has a gene for antibiotic resistance
The plasmid is introduced into a bacterial cell and grown on the antibiotic
Only bacteria with the plasmid grow…the inserted gene is copied many times
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Promoter Coding Sequence Terminator
Cell specificityDevelopmental specificityStart transcription
Protein coding sequenceStop transcriptionMessage stability
Gene constructs can be moved into plants and the gene is expresseddriven by the promoter sequence
Anatomy of a Transgene
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Agricultural Biotechnology
PanaceaOr Pandora’s Box?
Genetically Modified Foods:
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Genetically Modified Organisms GMOHow is it done?
It is now possible to clone any gene
from any organism and move it into
plants
Agricultural Biotechnology
A Success Story:
Genetically Modified Salmon
Constitutive expression of rainbow trout growth factor
hormoneAll salmon shown are fourteen months old, those at the bottom
are the controls
Transgenic Atlantic Transgenic Atlantic salmon produced by salmon produced by Aqua Bounty Inc. Aqua Bounty Inc.
Growth rate, not Growth rate, not ultimate ultimate size is enhanced.size is enhanced.
Commercial Commercial production of Aqua production of Aqua Bounty salmon isBounty salmon isbeing reviewed by being reviewed by FDA. First transgenic FDA. First transgenic meat product. meat product.
Would you order genetically modified salmon at a restaurant if you also had a choice of wild salmon?
(A) yes(B) no(C) doesn’t matter(D) undecided
Tools and Techniques Tools and Techniques used in Biotechnologyused in Biotechnology
Gene transfer from one organism to another is not new
Image of two species of bacteria transferring viralphage particles
Bacteria transfer genesto other bacteria and plants
Now in nature thereis another organismcapable of Transferring DNA: we call that organisma human being
Electro refers to the energy of
electricity. Phoresis, from the
Greek verb phoros, means
“to carry across.” Thus, gel
electrophoresis refers to the
technique in which molecules
are forced across a span of gel,
motivated by an electrical
current.
Gel Electrophoresis:the separation of molecules,DNA, RNA and proteinsby charge and size
Applications of Gel Electrophoresis
• DNA Fingerprinting
• DNA Recombinant Technology
• Forensics
• The Human Genome Project
DNA carries a net negative charge; it is negatively charged because the phosphates (red circles) that form the sugar-phosphate backbone of a DNA molecule have a negative charge.
As the separation process continues, the separation between the larger and smaller fragments increases.
• Molecular weight markers are often electrophoresed with DNA.• Molecular weight markers are usually a mixture of DNAs with known molecular weights.• Molecular weight markers are used to estimate the sizes of DNA fragments in a DNA sample.
The Techniques of The Techniques of Molecular BiotechnologyMolecular Biotechnology
Technology has created new Fields
DNA detection GenomicsDNA synthesis Bioinformatics
DNA sequencing Pharmacogenomics
DNA cloning Transgenics
Expression cassette Computationalconstruction Biology
RNA detection Population Genetics
Protein detection Proteomics
Techniques of Techniques of Molecular BiotechnologyMolecular Biotechnology
• Polymerase Chain Reaction
• Southern Blot Analysis• Northern Blot Analysis• Western Blot Analysis
• cDNA Library Construction
• DNA Sequencing
• Gene Isolation
• Gene Vector Construction
PCRPCR
PCRPCRThe Polymerase Chain Reaction
PCRPCRThe Polymerase Chain Reaction
You Leave a Piece of You Behind
PCR was used on the “BLUE DRESS”
and showed President Clinton's association with
Monica Lewinsky.
PCR is AmplificationNobel laureate in Chemistry, 1994
Polymerase Polymerase Chain ReactionChain Reaction
THERE ARE MILLIONS OF DIFFERENT GENES THERE ARE MILLIONS OF DIFFERENT GENES OR SEQUENCES WITHIN ANY DNA SAMPLE OR SEQUENCES WITHIN ANY DNA SAMPLE (BLOOD, TISSUE, PLANT, ETC.). (BLOOD, TISSUE, PLANT, ETC.). A SPECIFIC SEQUENCE IS SELECTED TO BE A SPECIFIC SEQUENCE IS SELECTED TO BE AMPLIFIED (RED ABOVE). THIS SEQUENCE CAN AMPLIFIED (RED ABOVE). THIS SEQUENCE CAN BE ANY GENE OF INTEREST OR A NON-CODING BE ANY GENE OF INTEREST OR A NON-CODING MARKER REGION OF DNA.MARKER REGION OF DNA.
IN ORDER TO COPY THE SEQUENCE OR GENE, A SHORT SEQUENCE ON EITHER SIDE OF THE SECTION MUST BE KNOWN. THIS REGION (BLUE ABOVE) WILL SERVE AS A PRIMER ATTACHMENT SITE TO COPY THE DNA TARGET SEGMENT.
IN ORDER TO AMPLIFY A SPECIFIC FRAGMENT OF IN ORDER TO AMPLIFY A SPECIFIC FRAGMENT OF DNA, SEVERAL THINGS ARE NEEDED, INCLUDING DNA, SEVERAL THINGS ARE NEEDED, INCLUDING PRIMERS AND DNA POLYMERASE.PRIMERS AND DNA POLYMERASE.
AN ENZYME WHICH COPIES DNA, PRIMERS ARE AN ENZYME WHICH COPIES DNA, PRIMERS ARE SHORT PIECES OF DNA OR RNA DESIGNED TO SHORT PIECES OF DNA OR RNA DESIGNED TO PAIR WITH GENOMIC DNA AT A SPECIFIC PAIR WITH GENOMIC DNA AT A SPECIFIC ATTACHMENT SITE FOR THE MAIN PURPOSE OF ATTACHMENT SITE FOR THE MAIN PURPOSE OF HELPING THE DNA POLYMERASE BIND AT THE HELPING THE DNA POLYMERASE BIND AT THE DESIRED SECTION. DESIRED SECTION.
NUCLEOSIDE TRIPHOSPHATES, THE BUILDING BLOCKS OF DNA ARE ALSO NEEDED.
EACH NUCLEOSIDE TRIPHOSPHATE CONSISTS OF:
A BASE (ADENINE, THYMINE, CYTOSINE OR GUANINE).
A SUGAR AND THREE PHOSPHATES.
WITHOUT A SHORT PIECE OF DNA(OR RNA) TO ATTACH TO, DNA POLYMERASE CAN NOT COPY A DNA STRAND.
PCR REQUIRES SEVERAL CYCLES OF AMPLIFICATION. EACH CYCLE CONSISTS OF
THREE TEMPERATURE CHANGES.
THE STARTING TEMPERATURE (95 C) SEPARATES THE DNA THE STARTING TEMPERATURE (95 C) SEPARATES THE DNA STRANDS.STRANDS.
A LOWERED TEMPERATURE (50-60 C) ALLOWS PRIMERS TO BIND TO A LOWERED TEMPERATURE (50-60 C) ALLOWS PRIMERS TO BIND TO COMPLEMENTARY SEQUENCES IN THE DNA. COMPLEMENTARY SEQUENCES IN THE DNA.
A SLIGHTLY HIGHER TEMPERATURE (72 C) ALLOWS DNA A SLIGHTLY HIGHER TEMPERATURE (72 C) ALLOWS DNA POLYMERASE TO ATTACH TO THE PRIMERS AND COPY THE DNA POLYMERASE TO ATTACH TO THE PRIMERS AND COPY THE DNA STRANDS (EXTENSION).STRANDS (EXTENSION).
DNA STRANDS ARE SEPARATED BY HEATING @ 94o C.
THE TEMPERATURE IS LOWERED TO 54oC TO ALLOW PRIMERS TO PAIR WITH COMPLEMENTARY DNA SEQUENCES.
MAKING NEW DNA MOLECULES:
DNA POLYMERASE ATTACHES TO THE PRIMERS @ 72 C.
DNA POLYMERASE ADDS NUCLEOSIDE TRIPHOSPHATES TO THE PRIMERS TO COPY THE DNA STRANDS.
COPYING IS COMPLETED FOR EACH STRAND.
THE PROCESS IS REPEATED IN THE NEXT CYCLE.
THE TEMPERATURE IS RAISED AGAIN TO SEPARATE
THE DNA STRANDS.
THE TEMPERATURE IS LOWERED TO ALLOW PRIMERS
TO ANNEAL. DNA POLYMERASE ATTACHES TO
THE PRIMERS AND DNA IS COPIED TO MAKE
4 STRANDS OF DNA.
The stability of TAQ Polymerase allows for this amplification process through the three temperature changes.
THE PROCESS OF COPYING DNA STRANDS IS REPEATED
32-35 TIMES. WITH EACH AMPLIFICATION CYCLE,
THE NUMBER OF COPIES OF THE DNA SEQUENCE IS
DOUBLED UNTIL MILLIONS OF COPIES HAVE BEEN MADE.
Issues in Biotechnology
A method used to copy small amounts of DNA many times over was invented by Dr. Kary Mullis in the 1980s and is called PCR. PCR stands for:
(A) Protein Chromosomal Replication(B) Polymerase Chain Reaction (C) Pipetman California Reaction(D) Peptide Catalytic Reactors (E) Polysaccharide Catalyst Repair
Forensic applications of DNA based technologies
• Fingerprinting OJ Simpson
• Identification
• Paternity
• Crime Solving
• World wide data base
Forensic Identification: Basic PrinciplesEach of us is genetically uniqueIf enough genetic variation is tested, each
of us can be uniquely identifiedDNA is found in nearly all cells (blood,
semen, hair, etc.)DNA from an evidentiary sample can be
matched with DNA from a suspect to implicate or exonerate
...atatatacaacttactaccatataccgattacgatcgaattataccgcggacgtagtaatgacgatgaagtaactatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatactacctaccagggaggagata...
“Short Tandem Repeat sequence”
STRs Are Used in Identity Testing
Laboratory PCR Instrument
INPUT:Sample DNA, PCR enzymes,primers, individual nucleotide building blocks (and maybe fluorescent labels)
OUTPUT: Specific DNA fragments amplified millions of times for easy visualization
With sizes that vary between individuals
After PCR, DNA Fragments are Separated on a Gel
PCR products for each sample
-
+
TIME
5
45
15
10
40
20
25
30
35
Minutes FragmentSize
100
180
120
110
170
130
140
150
160
DNA fragments move throughgel based on their size
Analysis of four different sections of the DNA
Identity Testing Using PCR
Possible conclusions:
A. Suspect 1 DNA was at the scene
B. Suspect 2 DNA was at the scene
C. Suspect 3 DNA was at the scene
D. None were at the scene
E. Multiple suspects were at the scene
F. Data is inconclusive
S = size standardsV = victim’s DNA 1 = suspect #1 blood2 = suspect #2 blood3 = suspect #3 bloodE = evidence #1 S = size standards
50
450
150
100
400
200
250
300
350
S V 1 2 3 E S
A complete match!
S = size standardsV = victim’s DNA 1 = suspect #1 blood2 = suspect #2 blood3 = suspect #3 bloodE = evidence #1 S = size standards
PCR was used on the “BLUE DRESS”
and showed President Clinton's association with
Monica Lewinsky.
Issues in Biotechnology
DNA tests exclude Martin from gun grip in Zimmerman case September 19, 2012
Another round of evidence was released Wednesday in the second-degree murder case against George Zimmerman, including forensic tests that show Zimmerman’s DNA was the only one that could be identified on the grip of the gun used to shoot 17-year-old Trayvon Martin. Zimmerman’s DNA also was identified on the gun’s holster. The tests were inconclusive as to whether Martin’s DNA was on the gun’s holster.
Congratulations!!!!!You have $100,000.00!!!!!!! To Invest in Biotech.
Issues in Biotechnology
Stock Project The idea is to select five biotechnology companies and invest $100,000 (fictitiously, of course)
Issues in BiotechnologyStock ProjectYou can spread your money evenly across five companies (i.e.
$20,000 each) or not. For example, if a company is trading at $20/share you can purchase 1,000 shares for $20, 000. Look up companies on Google (e.g.: type Monsanto stock) and record their stock ticker designation (e.g.. MON of the New York Stock Exchange, NYSE). Observe their performance over the past year. Record current trading price per share and calculate how many shares you can buy. You must choose your companies and the number of shares. Submit a single page summary on these results. Toward the end of the course you will look up these same companies and determine the cost per share at that time. Calculate your losses or gains for each company and your total losses and gains. This project will be summarized at the end of the course in a one page summary of losses and gains.
Biotechnology Stocks Project
Congratulations!!!!!You have $100,000.00!!!!!!! To Invest in Biotech.
1. Select and Research five Biotech companies.2. Print and submit the current stock quote and for each company.3. Invest chosen amounts in each. Calculate the number of shares in each (Submit a One page summary).4. Contact company to receive investors information (Optional). 5. Monitor Stock during the course.6. Calculate gains and losses. Submit report with final exam.
Biotechnology and Industry
“In retrospect, recombinant-DNA may rank as the safest revolutionary technology ever developed.” - James D. Watson, Nobel Prize Winner, 1953
The Development ofBiotechnology is directly linked with Industry
The application of the biological sciences has moved from academia to the private sector
Driven by profits and the promise of profits
The distinction between Basic and Applied Science is blurred
Basic science is immediately applied in today’s biotech
Biotech Industry
Red vs Green companiespharmaceuticals vs agriculture (blood vs chlorophyll)
Information Sciences
GenomicsProteomicsPhenomicsBioinformaticsPopulation GeneticsClinical trials
The Role of Companies in the Development ofBiotechnology
Technology Development
Patents
Markets and Products
Market-driven Innovation
Genomics Companies
Celera
Incyte
Genome Therapeutics
Corp
Millenium
Paradigm Genetics
DuPont
Genaissance
Monsanto
TechnologyCompanies
Invitrogen Stratagene Qiagen Packard PE Biosystems Kairos BioRobotics
Biotechnology Industry
Advertisement of services in international journals
NATURE Science Nature Biotechnology
“Picks and shovels for the Gold Rush”
Innovative technologiesbecome biotech products
Big money in licenses
Commercialization ofbiotech products requiresFreedom to Operate (FTO)with all the
technologies used
And look at the ads!
Home Who We Are
Our Pledge Our Locations Company Leadership Company History Contact Us
Our Products Leading Brands Seeds & Traits Agricultural Productivity Product Pipeline Benefits of Our Products Technical & Safety Info
News & Media News Releases RSS & Email Subscriptions Monsanto in the News Key Contacts Calendar of Events Press Kit & Multimedia Executive Forum
Monsanto is an agricultural company. We apply innovation and technology to help farmers around the world be successful, produce healthier foods, better animal feeds and more fiber, while also reducing agriculture's impact on our environment. » View Biotechnology Videos Roundup RReady2Yield™ Soybeans — Another Step Closer to Farmers' FieldsRead More 2007 Farm Progress Show — “Road to Success” — Monsanto Technology ShowcaseRead More
Delta & Pine Land — Monsanto Company Begins Combining Delta & Pine Land BusinessRead More
Featured Story
$21 Million Data Center CompletedHigh-tech center will provide global IT support for Monsanto's growing data analysis needsRead More View All Featured Stories
Recent NewsMon, 17 Sep 2007 — Monsanto Increases Ongoing Earnings Per Share Guidance, Provides Free Cash Flow Guidance For Fiscal Year 2007 Fri, 14 Sep 2007 — Monsanto, Dow Agreement Paves the Way for Industry's First-Ever, Eight-Gene Stacked Offering in Corn Thu, 13 Sep 2007 — Monsanto to Provide Information to Iowa Attorney General Concerning Seed, Trait and Chemistry Business View All NewsJob Opportunities Monsanto Fund Scholarships News Releases Executive Speeches Financial Reports Our Locations Monsanto Gift Shop DEKALB WingWear MSDS & Labels Biotech Resources Biotech Knowledge Center Our Values, described in the Monsanto Pledge, guide our business decisions.Copyright © 2004-07 Monsanto CompanyContact Us | Sitemap | Legal Notice | Privacy Policy
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Our Pledge Business Conduct Corporate Governance Stewardship Corporate Giving Youth and Education Diversity
Investors Corporate Profile Stock Performance Presentations & Financial Reports Calendar of Events Contact & Shareholder Info
Careers Getting Started U.S. Job Opportunities International Job Opportunities Benefits & Rewards Career Development Diversity
» View Biotechnology Videos Roundup RReady2Yield™ Soybeans — Another Step Closer to Farmers’ FieldsRead More 2007 Farm Progress Show — “Road to Success” — Monsanto Technology ShowcaseRead More Delta & Pine Land — Monsanto Company Begins Combining Delta & Pine Land BusinessRead More
Featured Story
$21 Million Data Center CompletedHigh-tech center will provide global IT support for Monsanto’s growing data analysis needsRead More View All Featured Stories
Recent NewsMon, 17 Sep 2007 — Monsanto Increases Ongoing Earnings Per Share Guidance, Provides Free Cash Flow Guidance For Fiscal Year 2007 Fri, 14 Sep 2007 — Monsanto, Dow Agreement Paves the Way for Industry’s First-Ever, Eight-Gene Stacked Offering in Corn Thu, 13 Sep 2007 — Monsanto to Provide Information to Iowa Attorney General Concerning Seed, Trait and Chemistry Business
Stock Price
Monsanto (MON)
89.79 -0.66
Dow Jones (DJIA)
13,451.41-6.14Stock Chart | Annual Report
Stock Price
Stock Chart | Annual Report
Agricultural Biotechnology
Stock Price
Stock Chart | Annual Report
Stock Quote at: Sep 26 2012 12:22PM
Change 0.09 Vol 12,802,922
Day High 24.99 52 Wk. High 25.15
Day Low 24.79 52 Wk. Low 17.05
Open 24.91Mkt. Cap(Bil)
186.21
Prev Close 24.84
Stock Symbol: PFE Stock Exchange: NYSE
NYSE: PFE24.930.09 Pharmaceutical Biotechnology
Biotechnology Stock Project Example Report 1
Henrietta Lack Student’s Name PLS 190 Issues in Biotechnology Professor Albert Kausch
Due Date 10-03-2012
Biotechnology Company 1. Intel Corporation (INTC) $22.05 per share / 900 shares = $19,845 2. Amgen (AGN)$55.66 per share / 350 shares = $19,481 3. Pfizer (PFE) $14.78 per share / 1,500 shares = $22,170 4. Apple Inc. (AAPL) $413.81 per share / $18,621 5. General Electric (GE) $15.38 per share / 1,292 shares = $19,870.96 Total = $99,987.96
Biotechnology Stock Project Example Report 1
Student’s Name Jack StrawBCH 190 Issues in BiotechnologyProfessor Albert Kausch Date Due October 3, 2012
Biotechnology Company Amgem Inc. AMGN: 56.04 per share 356 shares for $20,000 Pfizer Inc PFE: 17.84 per shares 1121 shares for $20,000 Monsanto Co MON: 65.86 per share 303 shares for $20,000 Bio Rad Laboratories Inc BIO: 89.50 per share 223 shares for $20,000 Syngenta AG SVJ: 205.30 Euros per share 71 shares for 14567.49 Euro or $20,000
Total; $100,000
Biotechnology Companies:AstraZenecaActive MotifAventisAdventaCelera PEIncyte InvitrogenPfizerMerckSmith Kline BeechamNovartisMonsantoQiagenRocheParadigm Genetics
Chemicon InternationalTrevigenAvigenMetabasis TherapeuticsPintex PharmaceuticalsBioGenGarstPioneerDuPontPharmaciaBristol-MyersSquibbEli LillyCistem Molecular Corp.Operon Technologies
Biotechnology Companies:
DNX Transgenic SciencesChemdexMWGAG BIOTECHDouble TwistLarova BiochemieGreiner LabortechnikSungeneMolecular Devices Corp.Evotec BioSystems AGBIACORETIB MOLBIOLPE BiosystemsBruker Daltonics Inc.
Eppendorf ScientificCuragen CargillDow Agri SciencesDow Chemical Co.AmGenBayerDynalNoxxon PharmaScheringBoehringerRhein BiotechHyseqGenome Therapeutics
O sweet spontaneous earth how often have the doting fingers of purient philosophers pinched and poked thee, has the naughty thumb of science prodded thy beauty how often have religions taken thee upon their scraggy knees squeezing and buffeting thee that thou mightest conceive gods (but true to the incomparable couch of death thy rhythmic lover thou answerest them only with spring) e.e. cummings
For those who are interested in taking this For those who are interested in taking this course for college credit through the course for college credit through the
University of Rhode Island; University of Rhode Island; For more information please contact:For more information please contact:
[email protected]@gmail.com
CreditsCredits
Lectures by: Lectures by:
Edited by:Edited by:
Video Produced by:Video Produced by:
Thank You to The University of Rhode Island Thank You to The University of Rhode Island and all of the students of Issues in and all of the students of Issues in
Biotechnology over the yearsBiotechnology over the years
Dr. Albert KauschDr. Albert Kausch
Dr. Albert Kausch Dr. Albert Kausch and Kimberly Nelsonand Kimberly Nelson
Thaddeus WeaverThaddeus Weaver
