Transcript GPVEC 2008 Biotech part 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
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
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/
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/
Which bull would be the best sire?
Can you tell by their appearance?
Which bacteria is pathogenic to humans?
Can you tell by their appearance?
pathmicro.med.sc.edu
www.biology.iupui.edu
“Genetic Playbook”
www.petecarroll.com
http://www.kursus.kvl.dk/shares/vetgen/_Popgen/ge
netics/10/10/sld003.htm
All living organisms have DNA
Genome
Genome = all genetic material in a cell
Eukaryotes
Prokaryotes
Chromosomes
Chromosome
Plasmid/s
Deoxyribonucleic acid (DNA)
Deoxyribonucleic acid (DNA)
Nucleotides or bases
Adenine - A
Cytosine - C
Guanine - G
Thymine - T
Deoxyribonucleic acid (DNA)
Deoxyribonucleic acid (DNA)
Double Helix
Nucleotide or base
Major groove
Minor groove
Phosphate-deoxyribose
backbone
http://en.wikipedia.org/wiki/DNA
DNA Replication
Spontaneous mutation
Point mutation
Insertion
Deletion
1 error per 1,000 bacterial replication cycles
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”
L0029
L0028
tir
cesT
eae
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
Model Gene
Typically, cartoon renderings reflect only the single, “sense”
strand, but realize there is always also a complementary strand.
TATA (-30)
5’
ATG
TAA
INTRON
*
Promoter 5’-UT
E
I
E
AATAA
3’-UT
*
3’
Coding or
Sense strand
EXONS
(Exons contain protein coding sequence,
bacterial genes don’t have introns)
* - Transcriptional initiation /termination sites
TATA - Promoter element
TGA, TAA, TAG - Translational stop codon
ATG - Translational start codon
AATAA - Polyadenylation signal
Protein Biosynthesis
GGATCGGCTAGCTG……...CTACATAGCTAT
Gene
Transcription
GGAUCGGCUAAGCUAU
mRNA
Translation
Gly-Ser-AlA-…………..
Protein
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
DNA
Codon
mRNA
Codon
Encoded Amino Acid
The Genetic Code
Transfer RNA (tRNA)
Anti-codon
F.W. Nicholas, 1996, Introduction To
Veterinary Genetics. Oxford Univ.
Press
Protein
Amino(N)-terminus
Carboxyl(C)-terminus
DNA synthesized 5’-3’
Protein synthesized amino - carboxyl
Eukaryotic Protein Biosynthesis
TATA (-30)
5’
ATG
TAA
AATAA
*
3’
*
Promoter 5’-UT
Gene
Exon 1 Intron
Exon 2
*
5’ 5’-UT
mRNA
Exon 1---Exon 2
3’-UT
3’-UT
*
Transcription
(In nucleus)
AAAA
3’
Translation (@ ribosomes & tRNA)
(In cyctoplasm)
N-terminus
C-terminus
Pre-Protein
* - Transcriptional initiation /termination sites
TATA - Promoter element
ATG - Translational start codon
TAA, TGA, TAG - Translational stop codon
AATAA - Polyadenylation signal
Homework
Design you own gene
Double stranded DNA
Promoter element
Transcriptional initiation /termination sites
Translational start codon
Intron
Translational stop codon
Polyadenylation signal
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
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
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
DNA extraction
Important to have clean DNA for further experiments
“dirty” prep can have contaminates that inhibit enzymatic processes
Agarose gel electrophoresis
Cloning
http://www.accessexcellence.org/RC/VL/GG/plasmid.php
http://student.britannica.com/comptons/ar
t-90884/DNA-sequences-can-be-cut-intwo-ways?&articleTypeId=31
Restriction endonucleases
Enzymes that cuts double-stranded DNA following its specific
recognition of short nucleotide sequences, known as restriction
sites, in the DNA
Enzyme Source
Recognition
Cut
Sequence
5'GAATTC
5'---G
AATTC---3'
3'CTTAAG
3'---CTTAA
G---5'
EcoRI
Escherichia coli
EcoRV*
Escherichia coli
5'GATATC
3'CTATAG
5'---GAT
3'---CTA
BamHI
Bacillus
amyloliquefaciens
5'GGATCC
3'CCTAGG
5'---G
GATCC---3'
3'---CCTAG
G---5'
ATC---3'
TAG---5'
Ligase
An enzyme that can link together two DNA strands that have
single-strand breaks, i.e. DNA cut with a restriction endonuclease.
Cloning
http://www.accessexcellence.org/RC/VL/GG/plasmid.php
http://student.britannica.com/comptons/ar
t-90884/DNA-sequences-can-be-cut-intwo-ways?&articleTypeId=31
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 proteinencoding DNA content
Messenger RNA used as a
starting material
Messenger RNA reverse
transcribed into cDNA
Requires mRNA isolation
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
www.mun.ca/biology/scarr/PCR_sketch_3.gif
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.
Taq polymerase
Chien A, Edgar DB, Trela JM (1976).
"Deoxyribonucleic acid polymerase from the
extreme thermophile Thermus aquaticus". J.
Bact. 174: 1550–1557
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
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
New Sequencing technologies
Roche FLX 454
100 million bases per chip
$6,000
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 = $6,000
4 million bases
2 weeks from DNA extraction to sequence data
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
Heaton MP, Harhay GP, Bennett GL, Stone RT, Grosse WM, Casas E, Keele JW, Smith TP, ChitkoMcKown 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.
Association of disease traits –FPT
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.
0
5
10
15
20
25
30
35
SNPs in E. coli O157:H7
KS368
bovine
0
KS546
bovine
0
TX265
bovine
TX723
bovine
NE972
bovine
NE1370
bovine
CO50
bovine
TX376
bovine
Mass2
human
EDL 931
human
0
1271-84
human
0
EDL 933
human
0
CO147
bovine
0
CO713
bovine
0
IDPH31277
human
0
MARC611
bovine
0
NE1124
bovine
0
TW04863
bovine
WRRC1
human
NE1270
bovine
Sakai
human
TW05356
bovine
3526-87
human
100
Ability to predict those
isolates which can cause
disease in humans
100
91
89
87
100
0
78
100
97
94
95
B. Finlay
SNPs
Exon 1
gene
Exon 3
Exon 2
Exon 4
SNP
DNA trace
files
individual #1:
Aattaatgctt…
maternal chromosome
paternal chromosome …aatggtatcTattaatgctt…
…aatggtatc
MALDI-TOF
spectra
A
A/T
A/A
individual #2:
maternal chromosome …aatggtatcAattaatgctt…
paternal chromosome …aatggtatcAattaatgctt…
A/A
individual #3:
T/T
maternal chromosome …aatggtatcTattaatgctt…
paternal chromosome …aatggtatcTattaatgctt…
T
T/T
5100
5400
5700
Many different technologies for
SNP interrogation
Real-time PCR
Affymetrix
Biotrove
Illuminia
Sequenome
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 highstringency 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
Hybridization is the process of combining complementary, singlestranded nucleic acids into a single molecule.
Perfect...ATGCATGCATGCATGC...
::::::::::::::::
...TACGTACGTACGTACG*..
Imperfect...ATGCATGGGTGCATGC...
::::::: :::::::
...TACGTACGTACGTACG*..
G
T T
G G
...ATGCATG TGCATGC...
::::::: :::::::
...TACGTACGTACGTACG*..
DNA microarrays
mRNA expression
SNPs
Gene content
Imperfect-
http://www.bio.davidson.edu/Co
urses/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
Websites
http://www.dnai.org/
http://dynamicgene.dnalc.org/structure/structure.html
http://www.blackwellpublishing.com/trun/artwork/
Animations/cloningexp/cloningexp.html