level: Genomics and Transcriptomics 1
Questions and Answers List
Flashcards on the case on Genomics and Transcriptomics
level questions: Genomics and Transcriptomics 1
| Question | Answer |
|---|---|
| what are the cycles of PCR? | 0. initial denaturation: uncoiling the helical structure of DNA. 1. denaturation: separation of dsDNA strands by heat (95 degrees). 2. annealing: attachment of primers at 50 to 65 degrees. 3. extension: synthesis of new DNA by DNA polymerase and nucleoside triphosphates at 72 degrees. 4. final extension |
| how do the Taqman probe and SYBR green fluorophore work in qPCR? | taqman probe: under normal conditions, the probe is coiled up, meaning that fluorescein is near the quencher of the probe, limiting the fluorescent signal. when the target sequence is found, the probe will uncoil and bind to the sequence. as new DNA is created, the probe starts being degraded, which means that fluorescein is separated from the quencher and a fluorescent signal is shown. this is very specific. SYBR green: SYBR green dye binds to the minor groove of the DNA non-specifically and emits fluorescence. this provides information on each cycle of amplification and about the melting temperature. |
| what are the differences between PCR and qPCR? | PCR is qualitative, while qPCR is quantitative. if no detection method (e.g. gel electrophoresis) is done for PCR, there is no visual output. in qPCR, a fluorphore is added to the sample and the reaction is carried out in a thermal cycler which illuminates the sample with a light beam of a specific wavelength. in qPCR, the sequence copy number can be detected in real time. |
| what are the pros and cons of PCR? | pros: sensitive, specific, can analyse RNA and DNA, widely applicable, fast. cons: susceptible to contamination from other DNA or RNA sources, no novel information can be generated, requires expert knowlegde, base substitutions and other mutations can lead to inaccurate amplifications, qualitative results only. |
| what are the pros and cons of qPCR? | pros: faster than PCR, real-time monitoring, efficiency of reaction can be precisely calculated, quantitative analysis, high sensitivity and specificity, widely applicable, high throughput. cons: more expensive than PCR, quality and quantity can affect results, can produce false negatives if target sequence is only present at low concentrations, no novel information, limited to detection of DNA and RNA. |
| what is the purpose of RNA sequencing? | RNA seq is a sequencing technique that determines the presence of RNA in a biological sample and quantifies this amount. it can be used to analyse the transcriptome (the complete set of transcripts in a cell). RNA seq is used to give more insight into alternative splicing, mapping genes, mapping exon boundaries and to identify novel transcribed regions. |
| what is the process of RNA sequencing? | 1. RNA isolation from sample. 2. isolate specific RNA species you want to analyse, e.g. isolate miRNAs through size selection or other RNAs through poly-A selection. 3. conversion into cDNA. 4. add adaptors to both ends of cDNA fragments. 5. align the fragments with the reference genome. 6. classify reads as either exonic reads, junction reads and poly(A) end-reads. 7. the result is quantified by counting the number of reads that align to each gene using a computer program. |
| what are the pros and cons of RNA sequencing? | pros: high resolution (up to single bases), high throughput, low background noise, able to differentiate isoforms, able to distinguish allelic expression variants, required amount of RNA is low, can reveal SNPs or other mutations, can be used to identify new genomic sequences (novel information), can be used to map complex transcriptomes. cons: expensive (but relatively low cost for large mapping large transcriptomes), difficult to store, retrieve and process large amounts of data, time-consuming, reverse transcripton needed, complex to analyse. |
| what is RNA interference and what is its purpose? | RNAi is a phenomenon in which small pieces of RNA can shut down protein translation by binding to mRNAs that code for those proteins. it is a process ocurring naturally. its main application is gene silencing. this can be used to silence genes in certain diseases, to inibit viral RNA and prevent viral replication, to silence cancerous genes thereby preventing tumour growth or to enhance traits in crops and plants. it can also be used to create knock down models to identify functions of certain genes. |
| what is the procedure of RNA interference? | 1. the enzyme Dicer cleaves the long dsRNAs into short double-stranded fragments of 21-23 nucleotides, called small interfering RNAs (siRNAs). 2. each siRNAs is unwound into two single-stranded RNAs; the sense and antisense strand. 3. the sense strand is cleaved by the protein argonaute 2 (Ago2) and degraded, the antisense strand is incorporated into the RNA-induced silencing complex (RISC). 4. RISC then binds and degrades the target mRNA (that is complementary to the antisense ssRNA strand) through cleavage by Ago2. silencing of the gene can be confirmed by (q)PCR. |
| what is the principle of transitive RNA interference? | it is the movement of a silencing signal along a particular gene. it is the ability of the RNA interference to spread through certain organisms. it has been shown that this is possible in plants and the C. elegans. |
| what are the pros and cons of RNA interference? | pros: high specificity, low concentrations of dsRNA/siRNA needed, effective gene suppresion, widely applicable and therapeutic potential, siRNAs can be created exogenously and made to target specific mRNAs. cons: competition with endogenous RNAs (in case of exogenous administration), stimulation of the innate immune system, silencing of off-target genes which can create a toxic phenotype. |