Genome mapping and sequencing
24 important questions on Genome mapping and sequencing
Why is it not possible to just map the human genome by PCR and sequencing?
Which group of researchers found a method of sequencing without the known sequence?
What was the method the HUGO consortium used for sequencing?
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What is the advantage of using molecular cloning?
What kind of vectors can possibly be used for molecular cloning?
- Plasmids
- Phage / virus DNA
- YACs and BACs
What two kinds of enzymes are needed in the start of the process of molecular cloning?
- Restriction enzymes
- (T4) DNA ligase
What three sites do commercially available plasmids contain?
- An Origin of Replication (ORI)
- Selectable markers (for example, a gene for antibiotic resistence)
- A Multiple Cloning Site (MCS) (there the gene of interest is added, the site can be restricted by many enzymes)
How can you let plasmids replicate?
Where can restriction enzymes be found in nature?
How does a restriction enzyme work?
So the bacterium makes the restriction enzyme, but how does it protect its own DNA?
What is the function of DNA ligase?
How do selectable markers work?
- and certain conditions (for example, an ampicillin medium) can test if this gene is functional,
- so by adding ampicillin, you can test if the plasmid has taken up the DNA fragment.
Most plasmids and viral vectors can take up fragments up to 10 kb. What cloning vectors can take up larger fragments?
- Bacterial Artificial Chromosomes (BACs)
- P1 Artificial Chromosomes (PACs)
What cloning vectors are the most widely used in the lab, and why?
- more capacity means more efficiency
- they are easy to grow
What is a genomic library?
How was cloning used to sequence the human genome?
2. A BAC clone is selected from the library and again fragmentized
3. The fragments are sequenced
4. Teh sequenced fragments are assembled.
How does a (genomic) library look on an agar plate?
How is a restriction map created (fingerprinting)?
2. Clones with overlapping fragment lengths are identified
3. Contigs are combined
How does chromosome walking work?
2. A restriction map is created
3. A clone with the shortest overlap is identified
4. A probe is made from its end
5. Process is repeated
What is the 'minimum tiling path'?
What is the difference in sequencing method of HUGO and Celera genomics?
- HUGO: 'hierarchical shotgun sequencing': there are too many short DNA sequences to directly sequence, the Celera method gives more gaps.
How is a cDNA library made?
2. A DNA copy is made with reverse transcriptase
3. The RNA strand is degraded with RNase
4. A DNA strand (complementary to the DNA strand of step 2) is made by DNA polymerase (the RNA strand acts as a primer)
5. The result is a double-stranded cDNA
Why is there no need for the use of cloning in NGS?
- but for NGS you use adapters as a starting point for primers
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