Genetic Variation Flashcards
Population
Group of individuals belonging to same species in a defined geographic area and actually or potentially interbreed
Gene pool
Genetic info carried by members of a population
Population genes and environment
-A population that is well adapted to its environment may be considered highly homozygous since favorable alleles are likely to be present at high frequency
-Most populations, according to genetic evidence, shows that most populations have high degree of heterozygosity
Dots
Nucleotides that are the same as the consensus sequence
Letters
Nucleotude polymorphism
1000 Genomes Project
2008-2015
-Catalog 95% of common genetic variations carried by 7 billion people on earth
-2504 genomes sequenced from 26 populations
-Whole genome sequencing- low coverade, exome seq, microarray genotyping
Genetic variants in genome project
-88 million genetic variations identified
-87 million SNPS
-3.6 million INDELS (Short insertion and deletions)
-60.000 structural variants (copy number variation (CNV’s)
-Alu and Line-1 insertions
INDEL
Short insertion and deletion
CNV
copy number variation
Assumption about genetic variation
Assumption- wild type allele will be selected for and hence be more prevelant in the population
Neutral theory of molecular evolution
-Motoo Kimura 1968
-Mutations leading to detrimental amino acid substitutions= detrimental
-small fraction is favorable
-some mutations are neutral- functionally equivalent allele they replace
-Favouble mutations preserved while detrimental removed
-Frequencyof neutral alleles determined by mutation rates and random genetic drift (not natural selection)
-Some mutations at be fixed and others lost
-therefore diversity of alleles is a function of population size and number of neutral mutations
Exception of neutral theory
Some contribution of natural selection- adaption to environment- sickle-cell aneamia and malaria
Sanger sequencing current uses
Pre-determined DNA stretch with read length=800 bp
Now used for resequencing small DNA stretches
Next generation sequencing (NGS) advantages
Increased throughput of data per run
Reduction in cost
Sequence whole genome or transcriptomes
Huge amounts of DNA sequenced simultaneously
NGS Sample Preparation
-Different companies have different methods but all begin with DNA library prep
-DNA is isolated then fragmented into small pieces of appropriate length
-Sonication- sound energy used to shear DNA in solution
-Hydrodynamic shearing forced break DNA at random locations
-Fragments may have 5’ and 3’ overhangs
-Converted to blunt ends with polymerase and exonuclease
-Blunted fragments phosphorylated at 5’ end and A-tail at 3’ end
-Facilitates ligation of adaptor with T overhang
-Adaptir serves as primer binding site
NGS Amplifaction/ Cluster Generation
-After oligonucleotides are located to the ends of each fragment, fragments are amplified
-two methods for amplification
Emulsion PCR
Used by applied Biosystems (ABI SOLiD) platform
-individual DNA fragments are separated in a mixture of oil and water
-Aqueous solution containing PCR MasterMix, dNTPs and DNA are mixed with oil. Tiny droplets, each containing one DNA fragment created.
-Amplified in microreactors
Bridge Amplification
-Used by Illumina sequencing platforms
-Two-dimensional PCR of DNA fragments well separated and bound to glass slide in a flow cell
NGS Sequencing Reaction (illumina bridge amplification)
-In Illumina bridge amplication method, flow cell is a sealed glass device
-Reagents for sequencing can be passed through the glass channel
-Sequence is recorded during synthesis of complementary base
-ddNTPs not used in this NGS
-Each dNTP is labeled and emits a light signal
-ABI SOLiD system uses a technique called pyrosequencing-Involves the release of pyrophosphate when a base is incorporated
-signal is recorded after each base is incorporated with a CCD camera or electrical signal
