This document includes the summary (info slides + lesson notes) of lesson 1 of the course 'genome technology and applications'. Lesson 4 was simply an NGS introduction to the task. Here, you must give a group presentation about a certain NGS technique.
General info
- 4 professors
- Seminar on Next Genera3on Sequencing
o Prepara3on and a:endance is compulsory
o In small groups
o Prepare a small 10 minute presenta3on on a certain topic related to NGS
o Different topics foreseen
o Follow-up seminar with discussion
- Slides on BB
Cell-based DNA cloning
Principles of DNA cloning
- Developed in the 1970s
- Used a lot and also today
- 4 steps
- Early: cloning was used to amplify DNA. There was no PCR un3l the late 80s
1. In vitro construc.on of a recombinant DNA molecule
- Requires cuRng and pas3ng of DNA
o Restric3on endonucleases (cuRng)
o DNA ligase (pas3ng)
- Requires a replicon (= piece of DNA that makes independent replica3on possible )
o A piece of DNA that makes independent DNA replica3on possible
o Replicon is specific for a host
o Usually a construct called “vector” is used, containing many features used in the cloning
process.
§ A vector: usually a plasmid, but has engineered -> specific features that makes it
suitable
2. Transforma.on in a host (o<en a bacteria -> will replicate the DNA)
- Recombinant DNA molecule is introduced in a host cell
- Usually bacterium or yeast
o Easy to grown
o Fast reproduc3on
- For expression studies, cloning is o]en done in eukaryo3c cells (mammalian cells, insect cells,
see later in this chapter)
o For some research this is important. You bring your recombinant molecule in the
mammalian cells and you can study the cells.
- For expression studies, cloning in a bacterium usually precedes cloning in the host used for
the expression
o Cloning in bacteria will always need to be done. Before puRng the construct in the
mammalian cells you will make the construct in bacteria -> the finished product will be
cloned in mammalian cells
, 3. Selec.ve propaga.on of clones
- Cells are plated on agar
- Each individual cell forms a colony
- Each colony is a clone:
o All cells are iden3cal, and have the same ancestor cell
- 1 colony can be grown in liquid medium to obtain more cells
- A]er the transforma3on you can bring the bacteria on agar -> grown and form colony (all
cells in the colony are iden3cal). You can take one colony and growth it in liquid media
4. Isola.on of recombinant DNA clones
- The recombinant DNA is purified from the cells
- You can take out the construct a]er cloning
o You take one colony -> you put in a liquid medium -> growing it overnight at 37°C -> lyse
and purify
Further into it
1. Restric.on endonucleases
- CuRng
- Nomenclature: 1 le:er genus, 2 le:ers species, followed by number
o E.g. HaeIII: Hemophilus aegyp3cus
- We get our restric3on enzymes from bacteria
o Natural defence mechanism against bacteriophages
o DNA from bacteriophage enters the bacterium then it is cleaved
o There is a matching sequence specific DNA methylase
§ Methyla3on of the same recogni3on sequence
§ Bacterial DNA cannot be cut
è Restric3on endonucleases recognizes a specific DNA sequence -> will only cut that
sequence. It will methylate its own DNA and it will make the restric3on endonucleases that
way that its own DNA is not cut
o Type II RE will cut a specific recogni3on sequence
o Usually 4-8 bp
o Usually palindrome
- Cleavage
o On the symmetry axis: blunt ends
o Not on the symmetry axis : overhangs, s3cky ends, cohesive termini
The benefits of buying summaries with Stuvia:
Guaranteed quality through customer reviews
Stuvia customers have reviewed more than 700,000 summaries. This how you know that you are buying the best documents.
Quick and easy check-out
You can quickly pay through credit card or Stuvia-credit for the summaries. There is no membership needed.
Focus on what matters
Your fellow students write the study notes themselves, which is why the documents are always reliable and up-to-date. This ensures you quickly get to the core!
Frequently asked questions
What do I get when I buy this document?
You get a PDF, available immediately after your purchase. The purchased document is accessible anytime, anywhere and indefinitely through your profile.
Satisfaction guarantee: how does it work?
Our satisfaction guarantee ensures that you always find a study document that suits you well. You fill out a form, and our customer service team takes care of the rest.
Who am I buying these notes from?
Stuvia is a marketplace, so you are not buying this document from us, but from seller evagoormans. Stuvia facilitates payment to the seller.
Will I be stuck with a subscription?
No, you only buy these notes for $7.91. You're not tied to anything after your purchase.
