Summary of Genomics and Transcriptomics

Vak

Biomedical Approaches (MBS1002)

7 Documenten

Studenten deelden 7 documenten in dit vak

Universiteit

Maastricht University

Studiejaar: 2023/2024

Geüpload door:

Anonieme student

Dit document is geüpload door een student, net als jij, die anoniem wil blijven.

Maastricht University

Reacties

inloggen of registreren om een reactie te plaatsen.

Andere studenten bekeken ook

Gerelateerde documenten

Preview tekst

DNA sequencing

I. Goal II. Methodology A. Sanger DNA sequencing ● Up to 900 base pairs ● Primer extension with labeled ddNTPs (radioactive or fluorescent) ● Prior knowledge on target needed ● Difficult to detect variation in mixtures ● High accuracy + still in use for genotyping When it is a ddNTP, the reaction will stop since it is a modified dNTP. Thanks to a laser and detector that is going through the gel electrophoresis, you will obtain a chromatogram and therefore, the sequence.

Genomics and Transcriptomics

B. Next-generation DNA sequencing (NGS) (most used, gold standard)

Short fragments (150-300bp)
Parallel reaction on solid surface (bridge amplification) so millions of reads at the same time.
Sequencing by synthesis, fluorescence
High capacity (multiplexing), low cost, fast (hours compared to over ten years with Sanger), accurate (bit less than sanger but still very accurate) ● Library prep: ○ Fragmentation and size selection of dsDNA ○ Adaptors ligation (both ends with non-sequencing parts) ● Bridge-amplification (on a glass plates, to have a bigger signal) ● Template immobilization and spatial separation (clusters) ● Sequencing reactions and detection → Q how just one nucleotide at a time? ● Data analysis ○ Assemble NGS reads de novo ○ Align NGS reads to reference genome
Multiplexing possible
Cost effective
No prior knowledge of the genome required
Offers single-nucleotide resolution, detect related genes, alternative splicing, gene variants and SNPs
Higher reproducibility NGS disadvantages:
Infrastructure: sequencer, computer capacity and storage
Bioinformatics expertise
Cheap per base sequenced, very large capacity → supra-regional centralisation
Cost depending on how many reads you need. For a single one, Sanger is better because 1£ but 150 000 000 reads costs 1000£ with NGS. V. Applications Gold standard for molecular diagnostics → NGS Sanger: Non-compex, short, plasmid, genotype NGS: Variation, genomes Third generation: Phasing,modifications, no PCR VI. Innovative Applications

RNA sequencing

I. Goal RNA sequencing (RNA-seq) is a powerful and widely used technique for profiling the entire transcriptome of a biological sample, providing information about the types and abundances of RNA molecules present. II. Methodology Sample preparation: ● RNA extraction (mRNA, rRNA, tRNA, ncRNA) ○ Can be isolated or enriched ● RNA quality control (spectrophotometry and electrophoresis) ● RNA fragmentation (more manageable pieces) ● cDNA synthesis (with reverse transcriptase and random primers or oligo(dT) primers) ● Fragmented into smaller pieces ● Adaptor ligation ● Library preparation Sequencing:

The prepared library is loaded onto the sequencing platform, and the sequencing run generates millions of short sequence reads (fragments of cDNA) from the RNA molecules in the sample. Data analysis: Two ways: ● Aligning to a reference genome and de novo. ● Differentiation expression analysis - count the amount of mRNA that comes from each gene to know how much the transcription is. ● Visualization using matrixes, heatmaps, and clustering analyses. III. Examples IV. Pros and Cons V. Applications VI. Innovative Applications

Crispr/Cas

I. Goal Targeted genome editing based on a simplified version of the bacterial CRISPR/Cas antiviral defense system. II. Methodology Cas9 recognises PAM sequence that follows the DNA sequence target and cut. It is delivered with a guide RNA (=sgRNA= crRNA (gene specific)+tracrRNA (loading in Cas9)) so it is cut where we want.

Study gene of interest in model system
Identify region + KO/KI
Design efficient gRNA target sites (target-specific oligo with sgRNA enzyme mix and reaction mix, dna extension, transcription)
Combine sgRNA with Cas
Injection in fresh fertilized oocytes and check if variation there

A. PCR

The technique is based on the mechanisms of DNA replication, but performed in vitro. ● Denaturation: Double-stranded DNA is separated using heat. – the heat breaks hydrogen bonds between complementary DNA bases. ● Annealing: heat is lowered to allow the primers to attach. The exact temperature depends on the melting temperature of the primers. ● Elongation: Heat is increased again to optimal DNA synthesis temperature. DNA polymerase attaches nucleotides to the 3 ́end of each primer to synthesize a new daughter strand on each single strand, from 5 ́to 3 ́. (The DNA polymerase is often Taq polymerase from thermophilic bacteria)

Detection of amplification happens at the very end - after all the cycles (30 to 40 times). To visualize, stain the amplified DNA products with a chemical dye or label PCR primers with fluorescent dyes prior to PCR amplification and analyze thanks to agarose gel electrophoresis. B. qPCR Same principle as in PCR but gives quantitative results. → SYBR green-based or Probe-based: ● SYBR green-based : binds to newly synthesised double stranded DNA and fluoresces. ● Probe-based : probe specific to the target sequence is labelled with a reporter molecule and a quencher molecule. When nucleotides are inserted, the DNA polymerase displaces the reporter molecule from the probe resulting in fluorescence. We then get the Ct values (Cycle threshold which is the cycle number at which the fluorescence signal crosses a predetermined threshold. It is inversely proportional to the amount of target DNA. III. Examples IV. Pros and Cons PCR: Simple, rapid, highly sensitive BUT misleading results can easily appear because it is sensitive, prior sequence data needed for primers design, non-specificity of primers possible, incorrect nucleotides incorporated possible. qPCR: Fast and high, detect AND quantify, safer for contamination, multiplex option BUT expensive, PCR inhibitors in the sample can affect efficiency, specific primers design challenging, genomic dna contamination can alter gene expression, limited to known sequences amplification, analysis with standard curves complex. V. Applications PCR: Diagnose infections, crime investigation, genetic and disease research (cloning or sequencing, gene expression with RNA templates, genetic mutation/ polymorphisms/ variations associated with diseases detection)

qPCR: Gene expression analysis, pathogen detection, microbial load determination, genetic and epigenetic analysis, clinical diagnostics. VI. Innovative Applications

RNA interference

I. Goal RNA interference (RNAi) is a biological process in which RNA molecules inhibit gene expression or translation by neutralizing targeted mRNA molecules. It can be used as a molecular biology tool to study gene function or as a therapeutic approach to silence disease-causing genes. II. Methodology Short interfering RNA (siRNA) and microRNA (miRNA) III. Examples IV. Pros and Cons V. Applications Conserved process in cells: Role in development, immune response and silencing genes

Was dit document nuttig?