level: Genomics and Transcriptomics 2
Questions and Answers List
Flashcards on the lecture on the Genetic Code
level questions: Genomics and Transcriptomics 2
| Question | Answer |
|---|---|
| what is the method of Sanger sequencing? | in Sanger sequencing, the sequencing reaction takes place in four different tubes. each tube contains a primer, dNTPs and a DNA polymerase. each tube also contains a different radiolabelled dideoxy nucleotide (either ddATP, ddTTP, ddCTP or ddGTP) (at a very low concentration). when this ddNTPs is incorporated into the sequence, the reaction stop. the incorporation of the ddNTP is a random process. this means that the mixture will contain several DNA fragments of different lengths. when the length of the fragment is known (through gel electrophoresis), the location of the radiolabelled ddNTP can be estimated and the entire strand can be build. |
| what is the method of next generation sequencing (NGS)? | NGS is also called second generation sequencing. 1. isolate DNA and generate smaller fragments. 2. DNA fragments are coupled to different adaptors on both ends. 3. DNA fragment strands are then denatured. 4. separate DNA strands then bind with their adaptors to complementary sequences on the flow cell. 5. DNA is then amplified through PCR (while on the flow cell). 6. DNA is then denatured again and the strand that is not attached to the flow cell is washed away. 7. bridges are formed through the binding of the second adaptor to the cell. 8. bridges are amplified and both strands will stick to the flow cell. 9. bridge formation and bridge amplification is repeated several time to generate many copies. 10. once amplification is done, the reverse strand is cleaved and sequencing can start (the primer binds to the adaptors). 11. fluorescently labelled nucleotides are added and when the complementary nucleotide binds, it is excited by a laser and the fluorescent signal is obtained and the nucleotide is identified. |
| when would you perform Sanger sequencing? when would you perform next generation sequencing? | Sanger sequencing is nowadays only done when you know what you are expecting in a locus with very low diversity. in this case you have limited resources and only need a small result, and this will be cost effective. NGS is the gold standard for human DNA sequencing in diagnostics. it is low cost, very fast (compared to Sanger) and allows for multiplexing; different samples can be mixed and sequenced. |
| what are pros and cons of NGS? | pros: generates data from hundreds of sequences simultaneously, more sensitive to low frequency variations, little amount of DNA needed (because amplification is done), fast (human genome can be sequenced overnight), allows for multiplexing, cost effective, high reproducibility, a priori knowledgde of the genome is not required (adaptors do not bind complementary). cons: large infrastructure needed (sequencer, computer, storage, expertise), cheap per base but it generates so many bases that it is overall still expensive. |
| what are examples of third generation sequencing techniques? | PacBio: wells that have a DNA polymerase attached to their bottom are used. fluorescence nucleotides are added to the wells and the unknown DNA strand is extended by a polymerase. each time a fluorecent nucleotide is added, a signal is emitted. while the strand is elongated, you know the sequence. Oxford nanopore: native DNA is pulled through a very small channel, for each nucleotide that exists the channel, a little current flows through and is detected. each nucleotide will produce a different current, by which the nucleotide can be identified. |
| what are advantages and disadvantages of third generation sequencing techniques over NGS and Sanger sequencing? | advantages - no amplification is needed, which prevents PCR bias. - haplotying can be done; the determination of haplotypes (DNA variants of a single chromosome that are inherited together) from unordered DNA. - can generate very long sequences (200kb). disadvantages - higher error rate. - higher cost per kb compared to NGS. |
| what are the different forms of DNA damage; depurination, deamination and alkylation? | depurination: removal of the purine base (either guanine or adenine) of DNA deamination: the removal of the amino group of the DNA base alkylation: the addition of an alkyl group to the DNA, which has implications for DNA pairing (this the not the same as methylation, in which methyl groups are added to the sugar backbone of the DNA) |
| what are errors that can be made in the DNA? what are the results of these errors? what is their repair mechanism? | - replication errors > base mismatch and bulges > mismatch repair (MMR) - ROS > single strand breaks (SSB) > base excision repair (BER) - oxidation, alkylation, hydrolysis > single base damage > base excision repair - ionizing radiation > double strand breaks (DSB) > non homologous endjoining (NHEJ) and homology directed repair (HDR) - chemotherapeutics > interstrand crosslinks > non homologous endjoining (NHEJ), homology directed repair (HDR) and nucleotide excision repair (NER) - UV light, radicals > intrastrand crosslinks and bulky adducts > nucleotide excision repair (NER) |
| how are single-stranded breaks, double-stranded breaks, DNA adducts (crosslinks and oxidized bases) and mutations repaired? | SSBs: base excision repair (BER) DSBs: non homologous endjoining (NHEJ) or homologous recombination (HR) DNA adducts, crosslinks and oxidized bases: nucleotide excision repair (NER) mutation: mismatch repair (MMR) |
| what were the precursors of Crispr/Cas9 and what were their disadvantages? | meganucleases; only bind to a very specific DNA stretch and is only useful if this specific stretch is your gene of interest, cannot be targeted. zinc finger nucleases; can be targeted, but can only target specific sites (e.g. only 1000 genes can be targeted). TALEN; can be designed to any target site of the genome, but they are not very efficient and it takes a long time (weeks/months). |
| how does Crispr/Cas9 work? | 1. identify the sequence that has to be edited in the genome. 2. create a guide RNA that is complementary to the target sequence. 3. attach the guide RNA to Cas9 (a DNA cutting enzyme). 4. introduce the complex into the target cells. 5. the complex will recognise the target sequence and cut the DNA. 6. the cell will try and repair these breaks by non homologous endjoining. 7. NHEJ often makes mistakes, when these are not removed, the gene is knocked out. 8. when you want to create a knock in, you have to include a donor sequence that is complementary to the target site, except for the mutation that you want to introduce. for this process the repair mechanism of homologous recombination is needed. |
| what are the components of Crispr/Cas9? | Cas9: a nuclease that is originally isolated from S. pyrogenes, an effector protein. sgRNA: single guide RNA that arises because of the fusion of the gene specific crRNA sequence to the scaffold tracrRNA sequence. PAM: protospacer adjacent motif, a 2-6 basepair DNA sequence that immediately follows the DNA sequence that is targeted by Cas9. |
| what are next generations of Crispr/Cas9? | base editors: derivatives of Crispr/Cas9 that do not make DSBs but can cause certain point mutations depending on the enzymes that are included in the base editor. enzymes can be cytidine deaminase (C>G) and adenine deaminase (A>T). prime editing: does not make DSBs, but can produce any precise edit or insertion by using reverse transcription. a peg RNA is used and it can be programmable to everything you want to do. |