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I send you the main part of genetic exam “System of exclamation marks:
! - 50/60% chance it will appear on the exam
!! - 90%
!!! - definitely

!Homozygote – definition!
!Dominant homozygote – alleles AA!
!Recessive homozygote – alleles aa!
!Heterozygote – Aa A | | a!

Monogenic diseases: e.g. Cystic Fibrosis
Polygenic diseases: e.g. coronary artery disease

P – parents
F1 – first generation
F2 – second generation

!Test cross!
!Mendel's Laws!
Two-point cross
Codominance
Partial gene linkage
!Crossing Over!
Types of linkage analysis

!Genetic mapping in bacteria! !e.g. transduction!
!Genetic and physical mapping!

Levels of genome mapping:

Cytogenetic
Relative localization
Physical
Cloning

!To sequence DNA:
The molecule must be cut into many fragments and then an attempt is made to reassemble them into the original molecule. This is the so-called "shotgun" technique!

Problems associated with this method!

Other applications of genetic maps:
!- We observe a gene and find out what it does

We observe a trait and ask which gene is responsible for it
Identification of genes responsible for diseases

The first markers used for genetic mapping were markers:

!Genetic markers:
ADE2, CAN1, CUP1, CYH1, LEU2, SUC2, URA3!

Biological markers in humans:
Blood groups, the HLA-DRB1 gene has at least 1800 alleles, and HLA-B over 4200 alleles, visually observable traits

Physical mapping through direct DNA analysis

!Traits that are not genes and are used in mapping are called DNA markers, e.g. RFLP, SSLP (length polymorphisms)!

They must occur in at least 2 allelic forms (polymorphic)

RFLP analysis
Restriction mapping

Simple Sequence Length Polymorphisms (SSLP)
Minisatellites and microsatellites

Microsatellites are shorter and easier to study using PCR

Markers – Single Nucleotide Polymorphisms (SNPs)

!Humans have approximately 10 million SNPs in the genome!

Methods of SNP detection

Microarrays – a plate containing spots with attached short single-stranded DNA sequences

For example, 1A contains cytosine (C), therefore it genotypes guanine (G).
Meanwhile, 1B contains guanine (G) and genotypes cytosine (C).

If only 1A or 1B lights up, we know the individual is homozygous; if both light up, the individual is heterozygous.

Fluorescence in situ hybridization (FISH)

Physical mapping by transferring markers onto the DNA phase

STS – Sequence Tagged Site
EST is a type of STS

Localization of genes on chromosomes

Inheritance of sex-linked traits

Division of sequencing methods:

Chain termination method
Next-generation sequencing

Sequencing by chain termination method

The human genome has 3235 Mb – billions of base pairs

Advantages and limitations of chain termination sequencing

"The forgotten Maxam-Gilbert method" – using different chemicals, DNA strands were preferentially cleaved.

DNA pyrosequencing

Has a sensitivity of approximately 1–5%

Complementary nucleotide → fluorescence
No complementary nucleotide → no light

The stronger the signal, the more complementary nucleotides are present.

Next-generation sequencing (NGS):

2 main technologies:

Short reads from 100–400 bases dominate currently
Long reads in the thousands of bases

!NGS workflow:!

Data analysis – outline:

Mapping sequenced reads to the reference genome
Detecting differences between the sequenced DNA and the reference sequence
Aotating the significance of detected variants

We have 2 measurements in sequencing data:

Coverage – how much of the genome was covered
Sequencing depth – how deeply a region was covered

Software:
wANNOVAR, Ion Reporter, VarSome, Genomic Viewer

NGS strategies:
Whole genome, whole exome, amplicons, RNA transcriptome

Nanopores

There are technologies capable of sequencing a single cell.

Haemophilus influenzae was sequenced in 1995.

6.5 million species have been sequenced.

The human genome contains approximately 10 million SNPs.

Genome aotation

After sequencing, we must determine its function.

Gene Ontology (GO) nomenclature

!RNA!

Double-stranded RNA → cut into short siRNA fragments → binds to mRNA → mRNA is cleaved!

!Programmed nucleosomes!

!Gene overexpression!

!To understand gene function, we use !targeted mutagenesis! and the resulting phenotype!

!Genome-Wide Association Studies (GWAS)!

Genetic analysis of prokaryotic organisms and viruses. Structure of genomes in eukaryotic organisms.

Negative DNA supercoiling in prokaryotes

Negative superhelical structure

Vibrio cholerae has 2 circular DNA molecules.

!Difference between prokaryotic chromosomes, chromids, and plasmids!

Chromosome – located in the nucleoid, carries essential genes.
Chromid – uses a plasmid-type segregation system but also carries essential genes.

!Bacterial genomes have a compact genetic organization with very small gaps between genes. About 11% of human DNA is coding (1.5% in humans)!

!Transposons; sequences that do not undergo transposition are CRISPR and CAS(? – possibly written down incorrectly)!

!Operons!

The tryptophan operon contains 5 genes.

!The number of genes reflects the ecological niche a bacterium encounters (e.g. free-living bacteria are genetically more complex than parasitic ones)!

!The concept of the pangenome!

!Horizontal gene transfer!

!Metagenomics!

We can now identify bacteria by examining DNA found in the environment (e.g. soil or water) through sequencing!

!The microbiome in cancer treatment!

!Endosymbiotic theory!

!Mitochondrial genomes range from 5 to 1500 kb and contain 3–93 genes!

!Chloroplast genomes range from 60–525 kb and contain about 200 genes!”

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