Beef Cattle Production Management Series
(2008)
Introduction to Biotechnology
Part I
GPVECJuly 31st 2008Clay Center, NE
Jim Bono, PhDMicrobiologistUS Meat Animal Research CenterClay Center, NE
Overview of Parts I and II
Part IBiotechnology, GMOs, and Genetic Engineering
Molecular Genetics (DNA, RNA, and Proteins)
Part IIApplied Molecular Genetics
DNA extractionCloningDNA librariesPolymerase Chain Reaction (PCR)DNA sequencingSingle Nucleotide Polymorphism (SNP)Microarrays
Biotechnology is the application of scientific techniques to modify and improve plants, animals, and microorganisms to enhance their value.
Biotechnology
Agricultural biotechnology is the area of biotechnology involving applications to agriculture. Agricultural biotechnology has been practiced for a long time, as people have sought to improve agriculturally important organisms by selection and breeding. An example of traditional agricultural biotechnology is the development of disease-resistant wheat varieties by cross-breeding different wheat types until the desired disease resistance was present in a resulting new variety.
http://www.ctahr.hawaii.edu/gmo/intro/
In the 1970s, advances in the field of molecular biology provided scientists with the ability to readily transfer DNA — the chemical building blocks that specify the characteristics of living organisms - between more distantly related organisms. Today, this technology has reached a stage where scientists can take one or more specific genes from nearly any organism, including plants, animals, bacteria, or viruses, and introduce those genes into another organism. This technology is sometimes called genetic engineering. An organism that has been modified, or transformed, using modern biotechnology techniques of genetic exchange is referred to as a genetically modified organism (“GMO”).
Genetic Engineering & GMO
Roundup herbicide resistanceInsect resistance (Bacillus thuringiensis)Insulin productionEnviropig (low phosphorus manure - phytate - phytase)
http://www.ctahr.hawaii.edu/gmo/intro/
Which bull would be the best sire?
Can you tell by their appearance?
Which bacteria is pathogenic to humans?
pathmicro.med.sc.edu www.biology.iupui.edu
Can you tell by their appearance?
“Genetic Playbook”
www.petecarroll.com http://www.kursus.kvl.dk/shares/vetgen/_Popgen/genetics/10/10/sld003.htm
All living organisms have DNA
Genome = all genetic material in a cell
Chromosomes ChromosomePlasmid/s
Genome
Eukaryotes Prokaryotes
Deoxyribonucleic acid (DNA)
Deoxyribonucleic acid (DNA)
Nucleotides or bases
Adenine - ACytosine - CGuanine - GThymine - T
http://en.wikipedia.org/wiki/DNA
Deoxyribonucleic acid (DNA)
Nucleotide or base
Major groove
Minor groove
Phosphate-deoxyribose backbone
Double Helix
DNA Replication
Spontaneous mutation Point mutation Insertion Deletion
1 error per 1,000 bacterial replication cycles
A gene is a locatable region of genomic sequence, corresponding to a unit of inheritance, which is associated with regulatory regions, transcribed regions and/or other functional sequence regions.
A gene is a union of genomic sequences encoding a coherent set of potentially overlapping functional products".
A gene is often used to refer to an inheritable trait which is usually accompanied by a phenotype as in ("tall genes" or "bad genes")
Gene
Historically:“One gene, one Protein”
eaecesTtirL0028L0029
Gene content of various organisms
Species Number of genes
Mycoplasma genitalium 500
Streptococcus pneumoniae 2,300
Escherichia coli 4,400
Saccharomyces cerevisiae 5,800
Drosophila melanogaster 13,700
Caenorhabditis elegans 19,000
Sea urchin 23,300
Arabidopsis thaliana 25,500
Homo sapiens 27,000
Mus musculus 29,000
Oryza sativa 50,000
ATG TAA AATAATATA (-30)
I5’-UT E E 3’-UTPromoter* *5’ 3’
Model Gene
EXONS
INTRON
Coding orSense strand
TATA - Promoter element
ATG - Translational start codon
TGA, TAA, TAG - Translational stop codon
AATAA - Polyadenylation signal
* - Transcriptional initiation /termination sites
Typically, cartoon renderings reflect only the single, “sense” strand, but realize there is always also a complementary strand.
(Exons contain protein coding sequence, bacterial genes don’t have introns)
Protein Biosynthesis
GGATCGGCTAGCTG……...CTACATAGCTAT
GGAUCGGCUAAGCUAU
Gly-Ser-AlA-…………..
Gene
mRNA
Protein
Transcription
Translation
Transcription
Making a copy of the gene that can be used for translation
Protect the DNA
Uracil (U) instead of Thymine (T)
RNA polymerase reads the nucleotide sequence of the gene and makes a single stranded messenger RNA (mRNA)
http://www.dnai.org/a/index.html
Translation
Process of making a protein from the mRNA Changing language from nucleotides to amino acids
Ribosome is responsible for reading the mRNA and making the protein
Translational start – ATG
Translational stop – TAA, TGA, TAG
3 nucleotides are called a codon
Each codon codes for a specific amino acid – 20 amino acids
http://www.dnai.org/a/index.html
The Genetic Code
DNACodon
mRNACodon Encoded Amino Acid
The Genetic Code
F.W. Nicholas, 1996, Introduction To Veterinary Genetics. Oxford Univ. Press
Transfer RNA (tRNA)
Anti-codon
Protein
Amino(N)-terminus
Carboxyl(C)-terminus
DNA synthesized 5’-3’Protein synthesized amino - carboxyl
Eukaryotic Protein Biosynthesis
ATG TAA AATAATATA (-30)
Intron5’-UT Exon 1 Exon 2 3’-UTPromoter
TATA - Promoter element
ATG - Translational start codon
TAA, TGA, TAG - Translational stop codon
AATAA - Polyadenylation signal
5’-UT Exon 1---Exon 2 3’-UT
mRNA
Gene Transcription
AAAA
Pre-Protein
Translation (@ ribosomes & tRNA)
*
* - Transcriptional initiation /termination sites
*
* *
5’ 3’
5’ 3’
N-terminus C-terminus
(In nucleus)
(In cyctoplasm)
Homework
Design you own gene
Promoter element
Translational start codon
Translational stop codon
Polyadenylation signal
Transcriptional initiation /termination sites
Intron
Double stranded DNA
Met Pro Ile Gly Asn tataagaagatctaggaaaggagagattt ATG CCT ATT GGT AACatattcttctagatcctttcctctctaaa TAC GGA TAA CCA TTG
Asn Val Leu Gly Stopcttggtcataatccc AAT GTG CTT GGT TAA gaagatctaatagaaccagtattaggg TTA CAC GAA CCA ATT cttctagattat
agggatgcatccctccctacgtaggg
Homework example
Legendtataa – Transcriptional initiation signaltaggaaaggagagattt – 5’ UTRATG – Translational startcttggtcataatccc – intronTAA – Translational terminationgaagatct – 3’ UTRaataa – polyadenylation signal
Beef Cattle Production Management Series
(2008)
Introduction to Biotechnology
Part II
GPVECJuly 31st 2008Clay Center, NE
Jim Bono, PhDMicrobiologistUS Meat Animal Research CenterClay Center, NE
Overview of Parts I and II
Part IBiotechnology, GMOs, and Genetic Engineering
Molecular Genetics (DNA, RNA, and Proteins)
Part IIApplied Molecular Genetics
DNA extractionCloningDNA librariesPolymerase Chain Reaction (PCR)DNA sequencingSingle Nucleotide Polymorphism (SNP)Microarrays
DNA extraction
Important to have clean DNA for further experiments “dirty” prep can have contaminates that inhibit enzymatic processes
Agarose gel electrophoresis
http://www.accessexcellence.org/RC/VL/GG/plasmid.php
http://student.britannica.com/comptons/art-90884/DNA-sequences-can-be-cut-in-two-ways?&articleTypeId=31
Cloning
Enzyme SourceRecognition Sequence
Cut
EcoRI Escherichia coli5'GAATTC 3'CTTAAG
5'---G AATTC---3' 3'---CTTAA G---5'
EcoRV* Escherichia coli5'GATATC 3'CTATAG
5'---GAT ATC---3' 3'---CTA TAG---5'
BamHIBacillus amyloliquefaciens
5'GGATCC 3'CCTAGG
5'---G GATCC---3' 3'---CCTAG G---5'
Restriction endonucleases
Enzymes that cuts double-stranded DNA following its specific recognition of short nucleotide sequences, known as restriction sites, in the DNA
Ligase
An enzyme that can link together two DNA strands that have single-strand breaks, i.e. DNA cut with a restriction endonuclease.
http://www.accessexcellence.org/RC/VL/GG/plasmid.php
http://student.britannica.com/comptons/art-90884/DNA-sequences-can-be-cut-in-two-ways?&articleTypeId=31
Cloning
DNA libraries
Genomic library: Contains entire DNA content of an organism Suitable for determining genomic DNA sequence Requires chromosomal DNA isolation
cDNA library: Contains entire protein- encoding DNA content Messenger RNA used as a starting material Messenger RNA reverse transcribed into cDNA Requires mRNA isolation
Polymerase Chain Reaction (PCR)
www.mun.ca/biology/scarr/PCR_sketch_3.gif
PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications.
DNA cloning for sequencingDNA-based phylogenyfunctional analysis of genesdiagnosis of hereditary diseasesidentification of genetic fingerprints (used in forensic sciences and paternity testing)detection and diagnosis of infectious diseases.
In 1989 Science Magazine named Taq polymerase its first "Molecule of the Year".
Kary Mullis received the Nobel Prize in 1993, the only one awarded for research performed at a biotechnology company.
Taq polymerase
Chien A, Edgar DB, Trela JM (1976). "Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus". J. Bact. 174: 1550–1557
http://www.yellowstone.net/geysers/thermalfeatures.htm
en.wikipedia.org
DNA sequencing
The process of determining the exact order of the nucleotides/bases (A, T, C, and G) that make up the DNA of an organism.
Gene number, exact locations, and functions Gene regulation DNA sequence organization Chromosomal structure and organization Noncoding DNA types, amount, distribution, information content, and functions Coordination of gene expression, protein synthesis, and post-translational events Interaction of proteins in complex molecular machines Predicted vs experimentally determined gene function Evolutionary conservation among organisms Protein conservation (structure and function) Proteomes (total protein content and function) in organisms Correlation of SNPs (single-base DNA variations among individuals) with health and disease Disease-susceptibility prediction based on gene sequence variation Genes involved in complex traits and multigene diseases Complex systems biology including microbial consortia useful for environmental restoration Developmental genetics, genomics
Roche FLX 454100 million bases per chip$6,0001 week from DNA extraction to sequence dataE. coli genome 5.5 million bases – a 454 run will give an 18x coverageHuman genome 3 billion base – 30 runs would give a 1X coverage
ABI 3730 (384 well plate)422 thousand bases per plate9 plates = $6,0004 million bases2 weeks from DNA extraction to sequence data
New Sequencing technologies
Single Nucleotide Polymorphism (SNP)
DNA sequence variation occurring when a single nucleotide - A, T, C, or G - in the genome (or other shared sequence) differs between members of a species (or between paired chromosomes in an individual).
Not all SNPs cause a phenotypic change
50K SNP chip – interrogates 50,000 SNP
Parentage
Association of disease traits –FPT
Heaton MP, Harhay GP, Bennett GL, Stone RT, Grosse WM, Casas E, Keele JW, Smith TP, Chitko-McKown CG, Laegreid WW. Selection and use of SNP markers for animal identification and paternity analysis in U.S. beef cattle. Mamm Genome. 2002 May;13(5):272-81.
Clawson ML, Heaton MP, Chitko-McKown CG, Fox JM, Smith TP, Snelling WM, Keele JW, Laegreid WW. Beta-2-microglobulin haplotypes in U.S. beef cattle and association with failure of passive transfer in newborn calves. Mamm Genome. 2004 Mar;15(3):227-36.
05101520253035
100
0
0
100
91
89
87
100
0
0
0
0
0
0
0
0
0
100
97
94
95
78
KS368
KS546
TX265
TX723
NE972
NE1370
CO50
TX376
Mass2
EDL 931
1271-84
EDL 933
CO147
CO713
IDPH31277
MARC611
NE1124
TW04863
WRRC1
NE1270
Sakai
TW05356
3526-87
bovine
bovine
bovine
bovine
bovine
bovine
bovine
bovine
human
human
human
human
bovine
bovine
human
bovine
bovine
bovine
human
bovine
human
bovine
human
SNPs in E. coli O157:H7
Ability to predict those isolates which can cause disease in humans
B. Finlay
paternal chromosomepaternal chromosomematernal chromosomematernal chromosome …aatggtatcTattaatgctt……aatggtatcTattaatgctt…
…aatggtatcTattaatgctt……aatggtatcTattaatgctt…
individual #3:individual #3:
individual #2:individual #2:
paternal chromosomepaternal chromosomematernal chromosomematernal chromosome …aatggtatcAattaatgctt……aatggtatcAattaatgctt…
…aatggtatcAattaatgctt……aatggtatcAattaatgctt…
Exon 3Exon 3Exon 1Exon 1 Exon 2Exon 2 Exon 4Exon 4
genegene SNPSNP
paternal chromosomepaternal chromosomematernal chromosomematernal chromosome …aatggtatcAattaatgctt……aatggtatcAattaatgctt…
…aatggtatcTattaatgctt……aatggtatcTattaatgctt…
individual #1:individual #1:
T/T
DNA trace files
DNA trace files
A/A
A/T
T/T
5100 5400 5700
A/A
MALDI-TOF spectra
MALDI-TOF spectra
A T
SNPs
Many different technologies for SNP interrogation
Biotrove
Affymetrix Illuminia Sequenome
Real-time PCR
DNA Microarrays
A high-throughput technology that consists of an arrayed series of thousands of microscopic spots of DNA oligonucleotides of a specific DNA sequence. This can be a short section of a gene or other DNA element that are used as probes to hybridize DNA or cDNA sample (called target) under high-stringency conditions. Probe-target hybridization is usually detected and quantified by fluorescence-based detection of fluorophore-labeled targets to determine relative abundance of nucleic acid sequences in the target.
Hybridization is the process of combining complementary, single-stranded nucleic acids into a single molecule.
Hybridization
...ATGCATGCATGCATGC...
...TACGTACGTACGTACG*.. ::::::::::::::::
Perfect-
...ATGCATGGGTGCATGC...
...TACGTACGTACGTACG*.. ::::::: :::::::
Imperfect-
...ATGCATG TGCATGC...
...TACGTACGTACGTACG*.. :::::::
Imperfect-
G GT TG
:::::::
mRNA expression SNPsGene content
DNA microarrays
http://www.bio.davidson.edu/Courses/genomics/chip/chip.html
Homework
Describe PCR in your own words and pictures
Describe a potential application for SNP genotyping in veterinary medicine or beef production
http://www.blackwellpublishing.com/trun/artwork/Animations/cloningexp/cloningexp.html
http://www.dnai.org/
http://dynamicgene.dnalc.org/structure/structure.html
Websites