Animal Genomics

and
Methods for Genotype
detection
Agricultural animal research has been immense successful over the past century in
developing technology and methodologies that have dramatically enhanced production
efficiency of beef, dairy, swine, poultry, sheep and aquaculture industries.
Molecular biology, genomics, transcriptomics, proteomics, metagenomics,
metabolomics (“omic” science) have changed the face of animal research in the last 15
years.
In 2005, draft genome sequence of chicken and cattle has been published. Several
genomes (honey bee, canine, bovine, swine, feline species) are now in progres.This
raises the possibility of understanding the “quantitative trait” (phenotypic variations)
of animals and more accurate and rapid animal improvement
Negative side effects: Animal well-being and longevity in production environments,
losses in reproductive efficiency, increased stress susceptibility, increased animal
waste issues and increased susceptibility to animal metabolic and infectious diseases
New challenges: Well-being of live stock and poultry in environmentally neutral
production systems
Strength of animal Genomics:
1. The value of biodiversity
Information from the genome and the effect of its variations on phenotype will help to
clarify the molecular basis of adaptation within populations that have been under
selection pressure for 10,000 years (How many quantitative trait loci for each trait, how
many genes for a QTL, how many alleles for each gene et)

2. Leading age of quantitative genetics

In breeding within animal populations reduces heterogeneity resulting in “linkage
disequilibria” generated by selection within the breeds are significantly higher than
they are between the breeds. Comparative genomics (with human) in such condition
can be used to isolate the functional sequence and position of the gene in human.

3. Genetic engineering of livestock

Designer milk (increased casein content for cheese production, lactose free milk), resistance
to infections in animal etc.
Central Dogma in Molecular Biology

Transcription Translation

DNA mRNA Protein

Reverse transcription Folding
Non coding
3-D structure
RNA:
Post translational
microRNA
modifications
Trafficking (NLS, NES)

Cellular Functions

Physiological
Physiologicalfunctions
functions
(Function as an organism/species)
The many levels
of complex biological systems Organism/
Disease
Tissue

Cell
Network
(pathway interaction)

Pathway
Protein-Protein
Protein-DNA
Protein
-- including
RNA
many genetic variants,
DNA in both health and disease
 Genome DNA sequencing

mRNA Transcriptome Micro array

Proteome 2-D Gel, Mass

Spec.

Structural Proteome X-ray diffraction,

NMR

Biological Function
 Steps for the genome sequencing

Cells ~ 3 billion nucleotides

Technology
a. Rodent cell lines containing specific human
Chromosomes chromosomes, b. Radiation hybrids (fragments of human
~50x106 to 250x106 chromosomes in the rodent cell lines c. Separation of
nucleotides chromosomes on the basis of sizes by fluorescence
activated cell sorter (FACS)

Yeast artificial chromosome

Chromosome specific libraries, Pulse field gel
(YACs), Bacterial artificial electrophoresis to separate large ragment of DNA
chromosomes (BACs), 50x103 to (~106 nucleotides), Vectors (YACs & BACs) for
2000x103 nucleotides cloning large fragment

Clone small fragment

Technological advancement for sequencing, thousands
~ 103 nucleotides Sequence per day to thousand sequence per second,
Fluorescence based automation in sequencing
Sequencing

Data base search for annotation (making

sense of the sequence), new tools for Post genome (sequence) era
functional analysis in „silco‟,
Bioinformatics). 3-D structure
 What is genome annotation? Process of adding biology information and
predictions of a genome sequence (not the truth but nice approximation of truth)

“WET LAB”
ATG CAG CCG CTG CAT……….
Data base search for gene prediction
“In Silico” (promoter, start, stop, sizable aa , >100:
signal sensor method, extended lengths of
sequence like exons, introns etc.: content
Predicted Function sensor) sequence homlogy with other
genes/sequence, protein structure, fold
prediction, fold, regulation etc.

Biochemical/Physical/Biological experiments to confirm the prediction

 Human Genome: 24 distinct chromosomes, 3x10 9 bases

Intergenic region/non coding

Gene, ~30x103
sequences /junk DNA, ~80%
Sattelite DNA
Minisatellite (Markers)
1 2 3 4
Microsatellite (Markers)

Proteins: combinations exon Intron

of amino acids (~1x105)
Transcription

translation

1 2 3 4
Interactions and Biological
functions
Micro array analysis of the transcriptome

Spotting the targets on solid surface

Label DNA/RNA with fluor
Hybridise to DNA spots on matrix
Detect bound DNA by scanning
Bioinformatics analysis of the genes (clustering etc)
Overview of Procedures for Microarray
Analysis of Tumor Samples

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Source: N Engl J Med, Vol 344 (8), 2001.

 What is mutation
Mutation and Polymorphisms

Mutation in general is heritable changes in DNA sequence, and

change produces detectable effect on a gene product.

Polymorphism = presence in the same population of two or more

alternative forms of a DNA sequence, with the most common
allele having a frequency of 99% or less (thus the rare allele
frequency is less than 1%). Any two individuals have a
polymorphic difference every 1,000-10,000 base pairs.
 Mutation Polymorphisms
CGC AGC GGC GAA CGC AGC GGC GAA
Arg---Ser---Gly---Glu Arg---Ser---Gly---Glu
CGC AGA GGC GAA
CGC AGA GGC GAA
CGC AGU GGC GAA
Arg---Arg---Gly---Glu Arg---Arg---Gly---Glu
Arg---Ser---Gly---Glu
Population Frequency < 0.01 Population Frequency > 0.01

Change in structure and Change in function (???)

function and structure (??????)
Single nucleotide polymorphisms (SNPs): The most abundant
variations in the genome

CGG TA C T T GA G GCT A Person3

Population Frequency > 1%

Autosomal Dominant

Autosomal recessive

Mendelian transmittance
 Types of Mutation/DNA variations

Chromosomal abnormalities (mainly in somatic changes in cancer:Bcr-Abl

translocation in CML)

Point mutation (miss-sense, non-sense, insertion/deletion, frame shift)

Ministaellite variations
Microsatellite Variations
Copy number Variations
Cytogenetics (chromosome banding, in situ hybridization, painting)
Southern Blot (chromosomal rearrangements, RFLP etc.)

Southern blots
PCR-based methods
Low throughput
Multiplex PCR (insertion/deletion), PCR-based RFLP, Allele specific Hybridization,
SSCP, Denature gradient gel electrophoresis (DGGE), Hetero-duplex analysis (HAD),
HAD-cleavage, microsatellite (length determination)
High throughput
DNA sequencing, microarray, Real time PCR, Comparative genome hybridization (CGH,
copy number variations, rearrangements, amplification/deletion of large segments)
Karyotype
Chromosome
Painting (CV)
 Comparative Genomic Hybridization (CGH):
Characterizing gene amplification and deletion

From Jonathan Pollack Lecture 2005

Strategies for mutation (Known) detection
 Basic methodologies

Hybridization
Dot blot, microarray, Taqman, Molecular Becon (Real time PCR)

Allele specific PCR

FRET, intercalating dye

Primer extension
SNAP-shot

Endonucleage
RFLP, Heteroduplex cleavage
PCR to amplify the target region of the genome
Nucleic Acid Hybridization Probes
(allele discrimination)
End-Labeling (detection)
RFLP-Southern blot
PCR-RFLP Typing
Allele-Specific Olignonucleotide (ASO) Dot Blot
Hybridization
• Dot blot: aqueous
soln of target DNA
denatured and
allowed to dry on
nitrocellulose
membrane,
• Single-stranded
labeled probe with
central mismatch
Hybridization Stringency
 SSCP: Denatured conformation (shape) of DNA molecules is dependent on the DNA
sequence, and the shape modifies the migration rate in electrophoresis

C
1 2 3 4 5 6
1 2 3

101bp

T
A B

Detection of DNA variation C-T (A762V) of PARP-1 gene.

A: PCR product of 101 bps. Lane 2 and 3 were the desired PCR products and Lane 3 contained no DNA control.
B: Detection of variant mobile bands by SSCP in 15% polyacrylamide silver stained gel. After validation by sequencing
the variant bands in Lane 1, 2, 3, 4, 5 and 6 were assigned for CC, CT, TT, TT, CT and TT genotypes respectively.
C: Sequencing of the PCR product. The 101bps PCR product was cloned in TA cloning vector and sequenced using
MegaBase 500 DNA sequencer. The upper chromatogram represented the C variant and the lower one represented the T
variant respectively.
 SSCP/RFLP

279 bps
1 2 3 4

160 bps
279 bps

119 bps

1 2 3 4 5 6

A B C

Detection of DNA variation G-C (R72P) in of p53 gene.

A: PCR product of 279 bps. Lane 1, 2, 3 were the desired PCR products and Lane 4 contains no DNA control.
B: Detection of variant mobile bands by SSCP in 12.5% polyacrylamide silver stained gel. After validation by
RFLP the variant bands in Lane1, 2 and 3 were validated for GG, CC, CC, GC, GC and GG genotypes.
C: RFLP patterns with BstUI digestionof the 279 bps PCR product. Lane U contained the undigested 279 bps
PCR product. G variant yielded two fragments on digestion with BstUI of the size 160 and 119 bps whereas the
C variant did not contain the RE site and yielded the undigested fragment of 279bps. Lane 1, 2, 3, 4, 5 and 6
were the genotypes GG, GG, CC, CC, GC and GG respectively.
 Heteroduplex analysis (HDA)
Figure 2 Endonuclease cleavage and exonuclease protection.
Figure 1. Formation of mismatched heteroduplexes
 Real-Time PCR (TaqMan)

Threshold; threshold
cycle

Litvak KJ. Allelic discrimination using fluorogenic probes and 5’ nuclease activity.
Genet Anal 1999
Allele-Specific PCR

• 3’ mismatch
• High stringency
• Certain mismatches
are more unstable
than others
Microsatellite Repeat Typing
Steps in DNA Sequencing and
Genotyping
Selecting part of genome to sequence or
genotype
Amplifying nucleic acid
Determine sequence of one or a succession
of nucleic acids:
Chemical reaction format
Read out format

Resequencing vs de novo sequencing

Dideoxynucleoside Sequencing
Fluorescent Labels
Dideoxynucleoside Sequencing
End-Labeling
Dideoxynucleoside Sequencing