Question

Biochemistry Tutorial: DNA Cloning

Answer 1

\({\bf{Overview:}}\)

in the context of DNA, a clone is a copy of a gene or gene segment

basic procedure of recombinant DNA/genetic engineering process:
1. obtain the desired DNA segment you want to clone w/ restriction enzymes or shearing
2. select a cloning vector, which is a segment of DNA that self-replicates. generally sourced from bacteria, yeast, or viruses
3. join the DNA segment w/ the cloning vector, typically w/ DNA ligase. result is recombinant DNA
4. transfer recombinant DNA to the host organism to be replicated using the host's internal replication mechanisms
5. select for the host cells that have taken up the recombinant DNA

Answer 2

\({\bf{Restriction:}}\) the process by which DNA is recognized and cleaved
restriction-modification system: process by which a host modifies (typically through methylation) sequences to be protected from restriction enzymes

three types of restriction endonuclease:
type I:
- large multisubunit
- has endonuclease + methylase capabilities
- cleaves DNA far from the recognition sequence (1000+BP)

type II:
- small
- does not use ATP
- catalyzes hydrolysis of phosphodiester bonds inside the recognition sequence
- short recognition sequences (4-6 BP)

type III:
- large multisubunit
- has endonuclease + methylase capabilities
- cleaves DNA close to recognition sequence (~25BP)

two cleaving types:
- sticky ends: cleavage results in 2-4 unpaired nucleotides at each end --> easier to ligate
- blunt ends: cleaveage does not result in unpaired nucleotides, instead goes straight across phosphodiester bonds

generally a recognition sequence occurs every 4^n base pairs (with n = the size of the sequence) assuming random distribution of nucleotides (generally not true in naturally occurring DNA)
partial digest process by which endonuclease reaction is stopped early to produce bigger fragments

Answer 3

\({\bf{Ligation:}}\) the process by which DNA fragments are joined together
- in general you can only ligate DNA fragments produced by the same restriction endonuclease
- catalyzed by DNA ligase, using ATP
- linkers: synthetic DNA fragments that connect the ends being ligated. called polylinkers if it has sequences for multiple endonucleases

Answer 4

\({\bf{Plasmids:}}\) circular DNA that replicates independently from its host
- contain sequences that help them replicate and express genes, making use of the host's natural processes
- may provide functions for the cell like antibiotic resistance
- contain origin of replication, and each plasma in the same chromosome in the cell must have its own oric or there will be competition for regulation

transformation: process by which plasmids are inserted into a cell (often bacterial, but can use other hosts, will go over this later)
- incubate the plasmid and the host cell at cold temp 0 degrees C
- heat shock to ~37C
- host cell will incorporate the plasmid (exact mechanism is beyond the scope of this course)
- alternative method: electroporation (applying a shock will make the cell membrane permeable for uptake of the plasmid)

after you incorporate the plasmid, you need some way to distinguish which hosts took up plasmid and which didn't. use markers

- selectable markers: allows cell to grow if they have the plasmid (positive selection) or kills cell if it took up the plasmid (negative selection)
- screenable markers: make the cell produce some detectable product, usu. a fluorescent molecule

Answer 5

\({\bf{Bacterial~Artifical~Chromosomes:}}\) vectors added to long segments of DNA
- stable orics, keep plasmid copies at 1-2 per cell
- par genes that influence distribution of genetic material at cellular division

alternative vectors: yeast artificial chromosomes (YACs)
- first treated as bacterial vector
- cleaved into vector arms
- partial digestion
- separate fragments w/ pulsed field gel electrophoresis
- ligation
- transform yeast cells
- selection based on selection factors

Answer 6

\({\bf{Gene~Expression:}}\)
- naturally occurring eukaryotic DNA is surrounded by sequences needed to express that DNA
- if you want to take a sequence from a eukaryote and express in a prokaryote, must insert bacterial regulatory sequences
- expression vectors: cloning vectors that also include regulatory sequences required for expression
- ideally, the promoter and the regulatory sequence should be near distinct restriction sites so genes are regulated from those regulatory elements

Answer 7

\({\bf{Hosts:}}\)
\({\bf{Bacteria:}}\)
- cheap, easy to grow, easy to store
- heterologous proteins may not fold properly or be modified/cleaved properly
- one method: fuse promoter and regulatory sequences of lac operon w/ the gene being studied, makes the gene expressed only when lactose is added. not perfect, because some product can still be expressed w/o lactose.
- alternatively, fuse to bacteriophage promoter and let the bacteriophage polymerase do the work instead

\({\bf{Yeast:}}\)
- most of the same advantages/disadvantages as bacteria but may be better for eukaryotic genes in terms of quantity, folding, etc.

\({\bf{Mammals:}}\)
- typically use a virus to insert the cloned gene
- expensive
- better at revealing how the gene/product might function in a person

Answer 8

this is getting a bit long so I'm going to make a part 2 of this

Source material is section 9.1 of Principles of Biochemistry 7th edition by Nelson, David L., and Cox, Michael M.