Beef Cattle Production Management Series

1
Beef Cattle Production Management Series
(2008) Introduction to Biotechnology Part I
Jim Bono PhD Microbiologist US Meat Animal
Research Center Clay Center NE
GPVEC July 31st 2008 Clay Center NE
2
Overview of Parts I and II
Part I Biotechnology GMOs and Genetic
Engineering Molecular Genetics (DNA RNA and
Proteins) Part II Applied Molecular
Genetics DNA extraction Cloning DNA
libraries Polymerase Chain Reaction (PCR) DNA
sequencing Single Nucleotide Polymorphism
(SNP) Microarrays
3
Biotechnology
Biotechnology is the application of scientific
techniques to modify and improve plants animals
and microorganisms to enhance their value.
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/
4
Genetic Engineering GMO
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).
Roundup herbicide resistance Insect resistance
(Bacillus thuringiensis) Insulin
production Enviropig (low phosphorus manure -
phytate - phytase)
http//www.ctahr.hawaii.edu/gmo/intro/
5
Which bull would be the best sire
Can you tell by their appearance
6
Which bacteria is pathogenic to humans
Can you tell by their appearance
pathmicro.med.sc.edu
www.biology.iupui.edu
7
Genetic Playbook
http//www.kursus.kvl.dk/shares/vetgen/_Popgen/gen
etics/10/10/sld003.htm
www.petecarroll.com
8
All living organisms have DNA
9
Genome
Genome all genetic material in a cell
Eukaryotes
Prokaryotes
Chromosomes
Chromosome Plasmid/s
10
Deoxyribonucleic acid (DNA)
11
Deoxyribonucleic acid (DNA)
Nucleotides or bases
Adenine - A Cytosine - C Guanine - G Thymine - T
12
Deoxyribonucleic acid (DNA)
13
Deoxyribonucleic acid (DNA)
Double Helix
Nucleotide or base Major groove Minor
groove Phosphate-deoxyribose backbone
http//en.wikipedia.org/wiki/DNA
14
DNA Replication
Spontaneous mutation Point mutation
Insertion Deletion 1 error per 1000
bacterial replication cycles
15
Gene
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)
Historically One gene one Protein
16
Gene content of various organisms
17
Model Gene
Typically cartoon renderings reflect only the
single sense strand but realize there is
always also a complementary strand.
ATG
TAA
AATAA
TATA (-30)
INTRON
3


5
3-UT
I
5-UT
E
E
Promoter
Coding or Sense strand
EXONS
(Exons contain protein coding sequence bacterial
genes dont have introns)
- Transcriptional initiation /termination
sites
TATA - Promoter element
TGA TAA TAG - Translational stop codon
AATAA - Polyadenylation signal
ATG - Translational start codon
18
Protein Biosynthesis
19
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
20
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
21
The Genetic Code
DNA Codon
mRNA Codon
Encoded Amino Acid
22
The Genetic Code
Transfer RNA (tRNA)
Anti-codon
F.W. Nicholas 1996 Introduction To Veterinary
Genetics. Oxford Univ. Press
23
Protein
Amino(N)-terminus
Carboxyl(C)-terminus
DNA synthesized 5-3 Protein synthesized amino -
carboxyl
24
Eukaryotic Protein Biosynthesis
ATG
TAA
AATAA
TATA (-30)
5
3


Intron
Promoter
5-UT
Exon 1
Exon 2
3-UT
Gene
Transcription
(In nucleus)


Exon 1---Exon 2
5-UT
3-UT
5
AAAA
3
mRNA
Translation (_at_ ribosomes tRNA)
(In cyctoplasm)
N-terminus
C-terminus
Pre-Protein
- Transcriptional initiation /termination
sites
TATA - Promoter element
TAA TGA TAG - Translational stop codon
AATAA - Polyadenylation signal
ATG - Translational start codon
25
Homework
Design you own gene
Double stranded DNA
Promoter element
Transcriptional initiation /termination sites
Translational start codon
Intron
Translational stop codon
Polyadenylation signal
26
Homework example
Met Pro Ile Gly Asn
tataagaagatctaggaaaggagagattt ATG CCT ATT GGT
AAC atattcttctagatcctttcctctctaaa TAC GGA TAA CCA
TTG Asn Val Leu Gly
Stop cttggtcataatccc AAT GTG CTT GGT TAA
gaagatctaata gaaccagtattaggg TTA CAC GAA CCA ATT
cttctagattat agggatgcatccc tccctacgtaggg
Legend tataa Transcriptional initiation
signal taggaaaggagagattt 5 UTR ATG
Translational start cttggtcataatccc intron TAA
Translational termination gaagatct 3
UTR aataa polyadenylation signal
27
Beef Cattle Production Management Series
(2008) Introduction to Biotechnology Part II
Jim Bono PhD Microbiologist US Meat Animal
Research Center Clay Center NE
GPVEC July 31st 2008 Clay Center NE
28
Overview of Parts I and II
Part I Biotechnology GMOs and Genetic
Engineering Molecular Genetics (DNA RNA and
Proteins) Part II Applied Molecular
Genetics DNA extraction Cloning DNA
libraries Polymerase Chain Reaction (PCR) DNA
sequencing Single Nucleotide Polymorphism
(SNP) Microarrays
29
DNA extraction
Important to have clean DNA for further
experiments dirty prep can have contaminates
that inhibit enzymatic processes
Agarose gel electrophoresis
30
Cloning
http//student.britannica.com/comptons/art-90884/D
NA-sequences-can-be-cut-in-two-waysarticleTypeId
31
http//www.accessexcellence.org/RC/VL/GG/plasmid.p
hp
31
Restriction endonucleases
Enzymes that cuts double-stranded DNA following
its specific recognition of short nucleotide
sequences known as restriction sites in the DNA
32
Ligase
An enzyme that can link together two DNA strands
that have single-strand breaks i.e. DNA cut with
a restriction endonuclease.
33
Cloning
http//student.britannica.com/comptons/art-90884/D
NA-sequences-can-be-cut-in-two-waysarticleTypeId
31
http//www.accessexcellence.org/RC/VL/GG/plasmid.p
hp
34
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
35
Polymerase Chain Reaction (PCR)
PCR is now a common and often indispensable
technique used in medical and biological research
labs for a variety of applications. DNA
cloning for sequencing DNA-based
phylogeny functional analysis of genes diagnosis
of hereditary diseases identification of genetic
fingerprints (used in forensic sciences and
paternity testing) detection and diagnosis of
infectious diseases.
www.mun.ca/biology/scarr/PCR_sketch_3.gif
36
Taq polymerase
Chien A Edgar DB Trela JM (1976).
Deoxyribonucleic acid polymerase from the
extreme thermophile Thermus aquaticus. J. Bact.
174 15501557
en.wikipedia.org
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.
http//www.yellowstone.net/geysers/thermalfeatures
.htm
37
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
38
New Sequencing technologies
Roche FLX 454 100 million bases per chip 6000 1
week from DNA extraction to sequence data E. coli
genome 5.5 million bases a 454 run will give an
18x coverage Human genome 3 billion base 30
runs would give a 1X coverage ABI 3730 (384
well plate) 422 thousand bases per plate 9 plates
6000 4 million bases 2 weeks from DNA
extraction to sequence data
39
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 50000
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 May13(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
Mar15(3)227-36.
40
SNPs in E. coli O157H7
10
15
20
25
30
35
0
5
bovine
100
bovine
0
Ability to predict those isolates which can cause
disease in humans
bovine
0
bovine
100
bovine
91
bovine
89
bovine
87
bovine
human
100
human
0
human
0
human
0
bovine
0
bovine
0
78
human
0
bovine
0
bovine
0
bovine
0
human
100
bovine
97
human
94
bovine
95
human
B. Finlay
41
SNPs
42
Many different technologies for SNP interrogation
Real-time PCR
Sequenome
Affymetrix
Illuminia
Biotrove
43
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.
44
Hybridization
Hybridization is the process of combining
complementary single-stranded nucleic acids into
a single molecule.
DNA microarrays
mRNA expression SNPs Gene content
http//www.bio.davidson.edu/Courses/genomics/chip/
chip.html
45
Homework
Describe PCR in your own words and pictures
Describe a potential application for SNP
genotyping in veterinary medicine or beef
production
46
Websites
http//www.dnai.org/
http//dynamicgene.dnalc.org/structure/structure.h
tml
http//www.blackwellpublishing.com/trun/artwork/An
imations/cloningexp/cloningexp.html