Lecture RR4. Dna cloning, experimental gener expression, introduction to genomes Flashcards
What is the benefit to using newer sequencing approaches?
They skip the PCR step ( Single molecule sequencing)
why do you need PCR when doing illumina sequencing?
You need it to detect the fluorescence
What does the DNA translocase do? And how does nanopore sequencing work
It’s a protein that will take DNA and make DNA move through it, it is inserted on a membrane containing different electrical voltages on both sides, so there is a current. When DNA moves through this current, it is possible to read it, creating ONT output (squiggles). It is possible to calibrate this sequencing for it to differentiate between the different bases going through the pore
How do u read the changes in voltage during nanopore sequencing
A small change in voltage = small molecule
A big change in voltage = a big molecule
What is the difference between Illumina sequencing, Sanger sequencing , and nanopore sequencing?
With Illumina and Sanger sequencing you can only sequence short strands of DNA, Nanopore sequencing on the other hand allows for much longer strands to be sequenced, which makes the assembly of long genomes MUCH easier!
What are the 3 main advantages of nanopore sequencing?
- Sequencing single molecules opens the possibility of studying new biological questions
- Very long reads minimize the need for genome assembly and allow mapping of repetitive sequences
- Portable, you can take it wherever you can take a laptop
What is recombinant DNA?
It is a DNA molecule made from 2 sources ( Vector [most of the time is a plasmid] + DNA fragment)
What is a plasmid? and what is its most important trait?
- most common vector used in recombinant DNA technologies
- ( MOST IMPORTANT ) plasmids are molecules of DNA that are autonomously replicated independently of chromosomes
- in the lab are circular dsDNA molecules
- There is a huge diversity of plasmids in nature
- Most plasmids have been described in bacteria, some in archaea and only a few in eukaryotes
what was the breakthrough that led to the use of plasmids in recombinant DNA?
the development of the use of restriction enzymes
what are restriction enzymes? and how do they create sticky ends?
enzymes that are going to cut the DNA, cut the phosphodiester bonds, usually in the lab they will cut it in two places that are symmetrically positioned. This will create “sticky ends” (Short single-stranded DNA strands that can be reannealed)
What is the ligase that is most commonly used for recombinant DNA?
T4 DNA ligase (T4 is a virus that infects bacteria)
What are the 3 important parts that plasmids used in labs need?
- Origin of replication, since it replicates independently of chromosomes :p , it allows the plasmid to be present in multiple copies.
- Resistance marker, it’s coding for the enzyme, its a gene that will be expressed in the cells
- Polylinker, a part of the sequence of the plasmid that contains multiple restriction sites ( 6 nucleotides in length)
What are the steps to transformation?
- treat the cells with calcium chloride, COMBINED with a heat shock, they can be introduced more easily into the cell
- that’s it. transformed cells will survive and the ones that didn’t transform will die (in the example of the antibiotic)
What does reverse transcription ( RT ) do? (Simple answer)
It takes RNA and turns it into cDNA (complementary DNA) with the help of a reverse transcriptase, an enzyme
what is the particularity of mRNA in eucaryotes? and how does it play in RT (reverse transcription)
They are processed in a way that adds a poly A tail, this tail can be used as a template for a primer (only containing Ts), then a reverse transcriptase can use this primer to copy the mRNA. at the end of the reaction we end up with a DNA molecule that is complementary (cDNA) to the RNA molecule.
