Sequencings
What are the steps of sequencing by synthesis?
Fragment DNA and add adaptors (primers) -> bind single stranded fragments to slide and amplify, creating clusters -> labeled nucleotides are excited by laser -> Capture image of first base pairs fluorescence. Repeat -> collect all the reads and align the data
What is somatic cell hybridization
Sendai virus to fuse the bovine and rodent tumor cells, only fused cells grow in the selective medium
Hybrid cells grow and divide, bovine chromosomes are progressively lost until one chromosome remains, collect DNA from each hybrid line, PCR amplify the cells DNA using your specific primers
What is linkage
Markers that are near on another are not inherited independently
Linkage = recombination rate is less than 50%
Probability of crossing-over is proportional to the distance separating the loci
The recombination rate can be used as a unit of chromosomal length - 1 cM = distance separating two loci exhibiting 1% recombination rate
1 cM corresponds to approximately 1 million basepairs
What is required for PCR + restriction fragment length polymorphism (RFLP)
Restriction endonucleases cut at specific sites creating fragments with blunt ends or overhangs
If SNP alters a restriction endonuclease recognition site
Gel Electrophoresis shows different lengths based on the restriction fragment
What is a LOD score?
Logarithm of the odds - Compares the likelihoods for linkage vs not linked
When the likelihood ratio is 1000:1 = 3
The log 10 of the likelihood ratio, the LOD score, equal to or greater than 3, then this is deemed proof of linkage
What is nanopore sequencing?
Electric current flows through the pore in the membrane
A single DNA molecule passes through, and disrupted current reads each nucleotide base
Long reads of about 100kb
A "motor protein" is used as a break to slow down how fast the molecule moves through the nanopore
What is in situ hybridization
Make a probe 100-10000 bp long that is complementary to a nucleotide sequence interest, label the probe
What are qualitative traits?
phenotypes are often discrete - double muscling gene, polled/horned gene, booroola gene, inverdale gene
less affected by environment
What are genome wide association studies (GWAS)
GWAS looks for association between a variant (SNP) and the trait of interest
DNA is hybridized to a SNP array
If one SNP is statistically more prevalent in one set of samples, it is associated with the trait
What are advantages of MAS
Faster selection process - detection is not dependent on the developmental stage
Ideal for traits with low heritability or difficult to assess
Can examine traits that can only be measured after selection has occured
What is array CGH?
Oligonucleotide probe = short single stranded pieces of DNA
Known probes from specific chromosome are bound to a slide, probes are space about 100kb, 50kb, or 10kb apart
Reference samples are differently labelled and hybridized to known probes on a slide
What is restriction mapping
Identifies where specific restriction enzyme sites are on small DNA molecules
What are polygenic traits
More than one gene and interacting factors
Trait is measured on a continuous scale
Genes that influence trait - QTL (quantitative trait loci) - meat tenderness, milk quality, weight, height
Microsatellite markers advantages and disadvantages
Advantages = Multi-allelic, found in all eukaryotes, technically easy, easy to compare between different analyses
Disadvantages - Not as abundant as SNPs not the causative mutation
What are disadvantages of MAS
Hitch hiking of linked undesired trait
It may be necessary to use larger population sizes or different genetic to develop reliable markers for MAS
What is physical mapping and types of methods?
Identifies genes and genetic markers in relation to specific chromosome locations
Methods = Somatic cell hybridization, in situ hybridization (radioisotope and fluorescence), restriction mapping, genome sequencing
What is linkage mapping
Genetic markers and a mapping population are required for linkage mapping
Genetic marker: DNA sequence used to identify genes on a genetic map
- SNP, Microsatellite, duplication/deletion
What is required for mapping a trait
polymorphic traits
Polymorphic markers - SNP and expansion-contraction polymorphisms
What is a PIC
What is a transgenic animal?
Is one that carries a foreign gene that has been deliberately inserted into its genome
DNA can be from the same species
Need not be genetically active
Foreign gene is heritable
What are Single nucleotide polymorphism DNA markers?
Simple substitution
Transition (pyrimidine substitution or purine substitution)
transversion (purine replaced by pyrimidine or vice versa)
What are microsatellite markers
Repetitive sequence
Small tandem repeats
Each animal will have two alleles
What is required for SNP detection?
Restriction fragment length polymorphism
KASP
SNP array
What is marker assisted selection (MAS)
Markers = DNA or RNA, polymorphic, linked to the gene you are selecting
Used in selection of superior traits
Genomic markers associated with QTLs (height, fertility, meat tenderness)
Association depends on the distance between the marker and the trait = linkage
MAS is beneficial when the traits are difficult and expensive to measure, with low heritability and/or recessive traits
Why are transgenic animals being produced?
study the genetic control of physiological systems
build genetic disease models
products for human therapeutic use
improve animal production traits
produce new products