Genetic Variation

Question 1

Population

Answer 1

Group of individuals belonging to same species in a defined geographic area and actually or potentially interbreed

Question 2

Gene pool

Answer 2

Genetic info carried by members of a population

Question 3

Population genes and environment

Answer 3

-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

Question 4

Dots

Answer 4

Nucleotides that are the same as the consensus sequence

Question 5

Letters

Answer 5

Nucleotude polymorphism

Question 6

1000 Genomes Project

Answer 6

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

Question 7

Genetic variants in genome project

Answer 7

-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

Question 8

INDEL

Answer 8

Short insertion and deletion

Question 9

CNV

Answer 9

copy number variation

Question 10

Assumption about genetic variation

Answer 10

Assumption- wild type allele will be selected for and hence be more prevelant in the population

Question 11

Neutral theory of molecular evolution

Answer 11

-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

Question 12

Exception of neutral theory

Answer 12

Some contribution of natural selection- adaption to environment- sickle-cell aneamia and malaria

Question 13

Sanger sequencing current uses

Answer 13

Pre-determined DNA stretch with read length=800 bp
Now used for resequencing small DNA stretches

Question 14

Next generation sequencing (NGS) advantages

Answer 14

Increased throughput of data per run
Reduction in cost
Sequence whole genome or transcriptomes
Huge amounts of DNA sequenced simultaneously

Question 15

NGS Sample Preparation

Answer 15

-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

Question 16

NGS Amplifaction/ Cluster Generation

Answer 16

-After oligonucleotides are located to the ends of each fragment, fragments are amplified
-two methods for amplification

Question 17

Emulsion PCR

Answer 17

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

Question 18

Bridge Amplification

Answer 18

-Used by Illumina sequencing platforms
-Two-dimensional PCR of DNA fragments well separated and bound to glass slide in a flow cell

Question 19

NGS Sequencing Reaction (illumina bridge amplification)

Answer 19

-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