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

Deoxyribonucleic acid (DNA)

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