Genomic Processes

• complete set of genetic information in an organism

• Genomics = study and analysis of genomes

• genome of the individual

• all genetic info inside a cell

• inherited from parents

• expression of genetype that is visible to people: eyecolor, size, skin, hair

• determinded by: genotype & environment

  = interaction of several alleles in different genes = found on different chromosomes

• different version of the same gene located at specific position on chromosome

• one allele from your mother and one from your father

• dominant: allele is expressed

• recessive: allele is masked

• Diploid organisms: 2 alleles (homo- or heterozygous)

• Two different alleles are expressed equally — neither one is dominant over the other.

• both alleles appear fully in the phenotype

• zB: AB blood type inheritance

  • A and B alleles are codominant.

  • If someone inherits A from one parent and B from the other → their blood type is AB.

  • Both A and B traits are fully expressed — not mixed or hidden.

• type of protein on outside of RBS

• determined by more than 1 gene

• zB: skin color, height, weight

• Phosphate

• Sugar

• Bases

  • Pyrimidines: Cytosin, Uracil, Thymine

  • Purines: Adenin, Guanin

• Pairing: T-A, G-C (G-U)

• Forward: 5-3 (Watson)

• Reverse: 3-5 (Crick)

• Chromatin: double stranded helical strct.

• DNA complexes with Histones = Nucleosome formation (consists of 8 Histone proteins (DNA wrapped around 1,65 times))

• Chromatosome: Nucleosome + H1 Histone, folds up to produce 30nm fiber

• Formation of loops (diameter 300nm)

• Fibers (compressed and folded into 250nm wide Fiber)

• tight coiling = Chromatid of chromosome

• type of protein found in the cell nucleus

• organizing and packaging DNA

• gene regulation (influence whether genes are turned on or off) = methylation

• present different amino acids:

  • Lysine

  • Arginine

• Phosphate groups = negatively charged

• AA = positively charged

  -> condense wrapping of DNA around histones

• introduction of Methyl functional group to Arg / Lys of Histone tail

• catalyzed by: Histone methyl transferase enzyme

• Methylated: 2x Arg, 3x Lys

Mitosis:

• Prophase

• Metaphase

• Anaphase

• Telophase

where cell duplicates ALL contents (incl. chromosome)

Result: 2 identical daughter cells

• G1: cell growth

• S: DNA Replication

• G2

• Mitosis: cell division

• Cytokinesis

Interphase: G1, S, G2

• Tradt. breeding

• Mutagenesis

• RNA interference

• Transgenics

• Gene editing

• green fluorescent protein

• protein in jellyfish

• exhibits green fluorescence when exposed to light

• has 238 AA

• Marker Protein to study developmental processes and metastasis in cancer

• 5000 - 8000

• defined as herited conditions arising from mutations on single gene

• affect 6%

• zB:

  • diastorphic dyplasia

  • nonsyndromic deafness

• Human genome sequencing

• Disease diagnostics

• Personalized medicine

• Forensic genetics

= Determining the complete human genome for the first time

Goal:

• create a "reference blueprint" of the human genome that future studies can rely on.

• built using DNA from multiple individuals to provide a complete and general representation of the human genome.

Features:

• Extremely complex and expensive (e.g., the Human Genome Project took over 10 years and billions of dollars).

• Serves as a comparison standard for sequencing individual genomes.

• The result is the "Human Reference Genome", which researchers worldwide use as a foundation.

• Determining the individual genome of a specific person

• for medical purposes or ancestry analysis, identify differences (compare to human reference System)

• Quick and affordable = NGS

• Whole genome sequencing WGS

• Exome sequencing Exome-Seq

• RNA sequencing RNA-Seq

• Methylation sequencing Methyl-Seq

• silent

• missense

• nonsense

1) Fusion by structural rearrangement

• translocations

• inversions

• deletions

• insertions

2) Fusion by transcription or splicing

• transcription read through

• mRNA trans-splicing

• cis splicing

1. Fragmentierung der DNA

   • Mechanisch oder enzymatisch → DNA wird in kleine Stücke zerteilt

2. Adapter hinzufügen

   • Spezielle Sequenzen werden angehängt, damit der Sequencer die Fragmente erkennt

3. Library Amplification

   • Verstärkung der DNA, damit das Signal beim Sequenzieren stark genug ist Methoden:

   • PCR (klassisch)

   • Emulsions-PCR (z. B. für Ion Torrent)

   • Bridge-PCR (Illumina)

   • Lineare Amplifikation (PacBio)

4. Größenselektion

   • Nur Fragmente einer bestimmten Länge werden weiterverwendet

5. Library Quality Control

   • z. B. mit TapeStation → prüft Fragmentgröße und Qualität

• TruSeq PCR free library preparation kit

• TruSeq Nano DNA library prep kit

• TruSeq stranded total RNA kit

• TruSeq DNA methylation kit

• Nextera DNA library prep kit

• Nextera rapid capture exome kit

1. Human genomes are 99.5% identical

2. Genetic variation arises from:

   • SNPs (Single Nucleotide Polymorphisms)

   • Indels (Insertions/Deletions)

   • Minisatellites & Microsatellites (also known as STRs)

Application: DNA Fingerprinting

• Especially useful in forensic science

• STRs are used to identify individuals

• STR profiling using Sanger sequencing

• Comparison with national DNA databases (e.g. UK, Spain, Germany)

• EDNAP = European DNA Profiling Group

1. large international project started in 2006

2. Sequenced over 20,000 samples across 33 cancer types

3. Aim: Understand the molecular basis of cancer and discover new biomarkers

Tools:

• TCIA, SurvNet, TANRIC, FASMIC

Applications:

• Tumor subtyping

• Personalized treatment

• Risk assessment

• Goal: Analyze cell-to-cell variation (e.g. within tumors)

Applications:

• Tumor heterogeneity

• Tumor evolution (e.g. during metastasis)

Isolation Methods:

• FACS, DEPArray, CellRaft

➤ Single-Cell DNA Amplification:

• MDA (Phi29 polymerase, high fidelity)

• DOP-PCR, MALBAC → alternative methods

• Challenges: ADO (Allelic Dropout), error rates, non-uniform coverage

• Technique for folding DNA into nanoscale structures

• Uses: long single-stranded DNA + short "staple strands"

Applications:

• Nano-containers

• Drug delivery

• Molecular sensors

• sample collection: from blood, saliva, tissue; goal: collect cells that contain DNA

• DNA extraction: isolate pure DNA from cell + remove proteins, other cellular material

• DNA fragmentation: Genome is too big = break DNA into smaller pieces (200-600 BP)

• Library perparation: add adapters to DNA fragments for recognition by sequencer (short known sequences)

• Sequencing: run fragments through sequencing machine (Illumination, Nanopore, Biopac) - each base sequence is read = output: 1000s of short sequence reads

• Data processing: quality controll + sequence allignment

• Data analysis: identify invariants + analyze genes, mutations, functl. elements

• DNA = natural data storage:

  • long lifespan

  • ultra compact

  • energy efficient

• DNA encoding: converting binary code (0,1) into bases (A,T,C,G)

• key components:

  • data

  • encoding system

  • writer (synthesizer)

  • reader (sequencer)

• human genome sequencing

• disease diagnosis

• personalized medicine

• forensic identification

• 1990 - 2003

• first full sequence of human DNA (3.1 billion BP)

• clone based & shotgun sequencing methods

• discovery of around 30.000 human genes

• Melting T°C:

  • T°C at which primer has dissociated from DNA template by half

  • 55 - 65°C

• Length & specifity:

  • long enough to anneal target region

  • not too long to form second structures

  • optimal: 18-24 nucleotides

  • avoid regions with: high sequence similarity / repititive DNA

• Secondary structures:

  • hairpin loops & self complementary sequences = can interfere with Primer Binding & amplification

• GC content:

  • GC content must be balanced

  • GC: 40-60%

  • target DNA content: 30-70%

  • GC clamp: GC within last 5 bases of 3’ end = helps to promote specific binding

• always consider: mutations & SNPs (=single nucleotide polymorphisms)

• very accurate

• slow + expensive

• for shorter sequences (1000 bp)

• dideoxy technique

• components:

  • DNA template

  • Primer

  • Polymerase

  • dNTPs (DNA building blocks)

  • ddNTPs (stop bases)

• Copy of DNA:

  • like PCR but add: special stop bases (ddNTPs)

  • mostly added: dNTPs = DNA grows

  • sometimes: ddNTPs = DNA immediatly stops growing

  • add them for all bases (each has different color): dATP, dTTP, dCTP, dGTP

• Many fragments are formed:

  • we get 1000s of fragments of different length = each fragment ends with specific base (where stop base was added)

• Sorting by length:

  • using gel-electrophoresis: shortest fragments move fastest!

  -> what color appears at each position (length) = tells us which base was at that position in original sequence

• knowing order in gene / genome for:

  • what gene it is

  • what protein it codes for

  • wheter theres a mutation

  • how to diagnose genetic diseases

  • how to design personalized treatments

• sequencing by synthesis (most common method)

• DNA fixed to flow cell surface

• Fluorescent nucleotides incorporated + detected by camera

• 13 billion reads per run

• Platforms: MiSeq, HiSeq, NovaSeq

• Nanopore sequencing

• produces long reads

• DNA pulled through protein nanopores

• changes in electrical current identifies bases

• Platform: MinION (portable device)

• Proton Detection Sequencing

• doesnt need light!

• detects pH changes from released H+ ions

• As DNA bases are added = pH increases = sensor measures voltage

• Platform: Ion Proton, PGM

• sequencing by ligation

• DNA read through fluorescently labelled ligation reaction

• very accurate

• complex data analysis & outdated

• emits light signal when nucleotide is incorporated

• Platform: Roche 454

• no longer in use

• to modify / enhance the characteristics of indivials or organisms

• can change:

  • one base pair

  • delete whole regions of DNA

  • introduce additional copies of a gene

  • combine genetic material from 2 different organisms

• delivery tool used to transport a desired gene into a target cell

• Use modified or weakened viruses.

• Viruses are naturally efficient gene carriers because they infect cells.

• zB: Adenoviruses, Lentiviruses (HIV-based), AAV (Adeno-associated virus)

Advantages:

• Very efficient at delivering genes into human cells.

• Can lead to long-term gene expression.

Disadvantages:

• Can trigger immune responses.

• Often complex and expensive to produce.

• use plasmids, liposomes, nanoparticles, or electroporation (electric pulses)

• No virus is involved

Advantages:

• Safer (less risk of immune reaction)

• Simpler and cheaper to produce

Disadvantages:

• less efficient at gene delivery

• Often short-term expression

• Promoter: initiates transcription of gene within a vector

• Origin of replication: needed to start replication process in host cell

• Cloning site: faciliates targeting of expressed protein to precise specific location (zB: periplasmic space of bacteria)

• AB resistance: for selection of vectors with genome of interest

• Epitope: antibody recognition of cell (some vector carry specific epitope sequences)

• Reporter genes: enable identification of plasmids that carry inserted DNA sequence

• Protein purification tags: faciliate purification process of expressed protein

• segment of DNA that contains the instructions to make a specific protein (sometimes RNA)