B Onichtchouk transcriptional regulatory network

DNA-binding proteins

bind to specific sequences -> DNA motifs

contain special DNA binding domain (homeobox) -> most evolutionary conserved part of the TF

can repress or activate transcription

Eukaryotes:

• TF targets more sequences in genome (shorter binding motive) -> mutiple TF regulate one gene (mediator complex)

• Chromatin structure !

Prokaryotes:

• TF targets one sequence (larger binding motive -> specificity) -> one TF can regulate gene expression

• polycistronic mRNA -> mutible genes controled by one operon

• regulation speed and no spatial or temporal separation of Transcrip and translation

  
  

A cic-regulatory element is a non-coding DNA region on the same strand / DNA molecule as the gene it controls, they way it regulates the DNA transcription can variy by type:

1. Promotors: transcription initiation RNA-polymerase binding site

2. enhancers: binding site for activators

3. silencers: binding site repressors

4. insulators: Block the interaction between enhancers and promoters or act as barriers to the spread of heterochromatin (compact)

ZRS = enhancer for Shh gene, essential for limp development

UAS = in yeast, Gal4/UAS system, transcriptional activator Gal4 binds to UAS

• DNAase footprint -> let the TF bind to the DNA and then digest it with nucleases where the protein was bound the DNA is intact and there will be no bands in the western blot

• Identification of in vivo TF binding sites by CHIP-seq -> peaks are TF binding sites (immunoprecipitated)

• yeast one hybrid = bait vector encoding DB of transcription factor of interest fused to GAL4 activation domain, prey vector: enhancer/silencer library + minimal promoter+ reporter/selection

• Illumina sequencing

  
  

p300 CHIP seq

—> DNA fragmentation and crosslinking

—> antibody against p300 (histone acetyltransferase, recruited to active enhancers)

—> IP and washing (e.g. magnetic beads or column), de-crosslinking

—> next generation sequencing

—> needs to be verified by reporter assay

Reporter assay

enhancer + minimal promoter + lacZ

high throughput method to identify enhancers

—> self-transcribing active regulatory regions

• DNA fragmentation, ligation of adapters

• library: DNA fragments are fused in front of minimal repoters and reporter

• introduction of constructs into e.g. yeast

• cells that are positive for reporter/ selection cassette contain construct with active enhancer

• sequence

used in NGS

• adapter ligation to DNA fragments

• immobilization of Fragments in flow cell via adapters

• denaturation + bridge formation

• amplification

• denaturation + amplification

• results in cluster formation

Solid phase bridge amplification is a technique commonly used in next-generation DNA sequencing technologies, particularly in Illumina's sequencing platforms. It involves the amplification of DNA fragments that have been immobilized on a solid surface, such as a glass slide, to create clusters of identical DNA molecules. Here’s a detailed explanation of the principle:

Principle of Solid Phase Bridge Amplification

1. Preparation and Immobilization:

   • The DNA sample is first fragmented into small pieces, and specific adapter sequences are ligated to the ends of these fragments. These adapters are complementary to sequences on the surface of the solid phase (e.g., a glass slide).

   • The solid surface is coated with two types of oligonucleotides (short DNA strands), which are complementary to the adapter sequences on the DNA fragments.

2. Hybridization:

   • The DNA fragments with adapters hybridize (bind) to the complementary oligonucleotides on the surface, anchoring the fragments to the solid phase.

3. Bridge Amplification:

   • The anchored DNA fragments are then denatured, separating the two strands.

   • One of the single strands loops over and hybridizes to a nearby complementary oligonucleotide, forming a bridge.

   • A polymerase enzyme then synthesizes a complementary strand, creating a double-stranded bridge.

4. Denaturation and Repetition:

   • The double-stranded bridge is denatured, separating into two single strands that remain attached to the surface at their respective ends.

   • This process of bridge formation, hybridization, and synthesis is repeated multiple times. Each cycle results in an exponential increase in the number of identical DNA molecules, forming dense clusters of DNA.

5. Cluster Formation:

   • Through multiple cycles of bridge amplification, millions of identical copies of each original DNA fragment are generated in discrete clusters on the surface.

   • Each cluster contains thousands of copies of a single DNA fragment, which can then be sequenced simultaneously.

Advantages of Solid Phase Bridge Amplification

• High Throughput: The technique allows for the simultaneous amplification of millions of DNA fragments in parallel, making it suitable for high-throughput sequencing applications.

• Spatial Separation: The solid phase provides spatial separation of DNA clusters, preventing cross-contamination between different DNA fragments.

• Efficiency: The amplification is highly efficient due to the proximity of the anchored oligonucleotides, which facilitates the formation of bridges and synthesis of complementary strands.

Applications

Solid phase bridge amplification is a critical step in Illumina’s sequencing-by-synthesis (SBS) technology. It is used in various applications, including whole-genome sequencing, targeted sequencing, RNA sequencing, and more.

By generating dense clusters of identical DNA fragments, solid phase bridge amplification ensures that there is sufficient signal for detection during the sequencing process, enabling accurate and comprehensive sequencing of complex genomes