Samenvatting - BMS75 - Advanced tools in molecular biology (BMS75) Summary

BMS75 – Advanced Tools in Molecular Biology




BMS75 – ADVANCED TOOLS IN
MOLECULAR BIOLOGY




Opleiding: Master Biomedical Sciences
Onderwijsinstelling: Radboudumc




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, BMS75 – Advanced Tools in Molecular Biology




Content
Designing molecular probes..............................................................................................................3
Basics of molecular (sub)cloning...........................................................................................................................3
Classical cloning...............................................................................................................................................4

Recombinases..................................................................................................................................................5

Restriction enzyme cloning.............................................................................................................................6

Primer design.........................................................................................................................................................6

Cell transfection................................................................................................................................6
Forms of material transfer....................................................................................................................................6
Transfection materials...........................................................................................................................................6
Transfection types.................................................................................................................................................7
Transfection methods...........................................................................................................................................7

CRISPR/Cas technologies...................................................................................................................8
Prime editing.......................................................................................................................................................10
Base editing.........................................................................................................................................................10

Functional protein analysis..............................................................................................................10
Epitope tags.........................................................................................................................................................10
Affinity tags..........................................................................................................................................................11
Purification....................................................................................................................................................11

Addition of functional proteins...........................................................................................................................11
Considerations for choosing a fusion tag and tag positioning............................................................................12

Cell selection and imaging molecular...............................................................................................12
Cell selection: stable pooles vs. clones...............................................................................................................12
Fluorescent protein vectors in cell- and molecular biology................................................................................13
Genetically encoded molecular probes: principles and applications.................................................................13
Single fluorescent protein-based probes......................................................................................................13

BiFC-based probes.........................................................................................................................................13

FRET-based probes........................................................................................................................................13




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, BMS75 – Advanced Tools in Molecular Biology


Designing molecular probes
Basics of molecular (sub)cloning
Cloning: Cutting a piece of DNA from one organism and inserting it into a ‘vector’ where it can be replicated by
a host organism (also called subcloning).

Recombinant DNA technology applies many different enzymes that modify DNA in one way or another:
 DNA polymerase: create DNA molecules by assembling nucleotides. Some polymerases have
proofreading activity (=DNA polymerase reads the newly added base before adding the next one, so a
correction can be made).
 Ligases: joins two ends of DNA strands with two requirements:
1. DNA ends need to match (base pair rules).
2. Ligase needs a 5'-phosphate group to be present to connect it to the 3'-OH group of the other
fragment.
 End-modification enzymes
 Klenow → makes 5’ overhangs blunt by filling in the missing nucleotides
 Shrimp alkaline phosphatase → removes phosphate group at the 5’ end of DNA strands,
preventing ligation of these fragments (chemically synthesized oligonucleotides lack 5’
phosphate groups)
 T4 polynucleotide kinase → adds (labelled) phosphate groups to the 5’ end of DNA strands,
enabling detection or ligation
 Nucleases (endonuclease and exonuclease): molecular scissors (endonucleases) that cut double
stranded DNA molecules at specific points and are found naturally in a wide variety of prokaryotes.
Host DNA is protected from cleavage by methylases which add methyl groups to adenine or cytosine
bases within the recognition site thereby modifying the site and protecting the DNA.

Isoschizomers: restriction enzymes that have the same recognition sequence as well as the same cleavage site.
Neoschizomers: restriction enzymes that have the same recognition sequence but cleave the DNA at a different
site within that sequence.

Plasmids are small, circular DNA molecules that are not part of the chromosomal DNA of bacteria.
They replicate independently, exploiting the host machinery, and confer selective advantage (e.g.
antibiotic resistance).
↪ Artificial plasmids are widely used as vectors in molecular cloning and are made non-
conjugating to prevent horizontal gene transfer.

A bacteriophage is a virus that infects and replicates within bacteria. Phages used for cloning are
phage λ and phage M13. λ phage vectors uses E. coli as host and have a typical phage structure:
head, tail and tail fibers. These vectors contain linear DNA with some unique cleavage sites
whereby foreign DNA with sizes of 5-11 kb may be inserted. M13 phages contain circular single-
stranded DNA and are the ideal vehicles for original Sanger sequencing.

A cosmid is a type of hybrid plasmid that contains a λ phage COS (cohesive end) sequence. It
was often used to clone large genomic DNA fragments (28-45 kbp). Unlike plasmids, they can
also be packaged in vitro into phage capsids.

An artificial chromosome is a synthetic, engineered chromosome that is designed to mimic the structure and
function of a natural chromosome found in living organisms. They can carry large amounts of genetic material
and are used as vectors to introduce new genetic information into cells. There are different types of artificial
chromosomes:
 Yeast artificial chromosomes (YACs): Based on the chromosome structure of yeast (a type of fungus),
YACs can carry very large fragments of DNA (100-1400 kbp). They can be circular (grows in bacteria)
and linear (multiplies in yeast). However, they are unstable.



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