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Chapter 9

CHY 204 Chapter Notes - Chapter 9: Plasmid, Molecular Cloning, Dna Ligase


Department
Chemistry
Course Code
CHY 204
Professor
Mario Estable
Chapter
9

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CHAPTER 9: DNA-BASED INFO TECHNOLOGIES
-Techniques for DNA cloning paved the way to the modern field of genomics & proteomics,
the study of genes & pro on the scale of whole cells & organisms. Paul Berg, Herbert Boyer
& Stanley Cohen developed the 1st techniques for recombinant DNA.
9.1 DNA cloning: The Basics
-A clone is an identical copy. DNA cloning involves separating a specific gene/DNA segment
form a larger chromosome, attaching it to a small molec of carrier DNA & then replicating
modified DNA 1000s or millions of times through both an inc in host cell #s & creation of
multiple copies of cloned DNA in each cell resulting selective amplification of a particular
gene/DNA segment. Cloning of DNA from any organism involves 5 general procedures:
1. Cutting DNA at precise locations: restriction endonucleases provide necessary
molecular scissors
2. Selecting a small mole of DNA capable of self-replications: cloning vectors which
is a deliver agent, typically plasmids or viral DNAs.
3. Joining the 2 DNA fragments covalently: DNA ligase linking cloning vector & DNA
to be cloned. Recombinant DNAs are composite DNA molec comprising covalently linked
segments from 2/more sources.
4. Moving recombinant DNA from test tube to host cell
5. Selecting/identifying host cells that contain recombinant DNA.
*E.coli is the most common host cell b/c its DNA metabolism is well understood,
many naturally occurring vectors are assoc w/ E.coli (plasmids & bacteriophages),
techniques are available for moving DNA from 1 bacterial cell to another.
-Restriction endonucleases & DNA ligase yield recombinant DNA: restriction
endonucleases/restriction enzymes recognize & cleave DNA at specific seq (recognition
seq/restriction sites) to generate a set of smaller fragments. DNA ligases joins the DNA
fragment to be cloned to a suitable cloning vector; the recombinant vector is introduced into
host cell which amplifies fragment via cell division. In the host cell’s DNA, the seq that would
be recognized by its own RE is protected from digestion by methylation of DNA catalyzed by
a specific DNA methylase. The RE+methylase=restriction-modification system. 3 types of
RE, Type I,II,III: Type I & III are generally large multisubunit complexes containing both
endonuclease & methylase activities; both req ATP. Type I RE cleave DNA at random sites
that can be more than 1000bp from the recog seq; Type III RE cleave DNA ~25bp from the
recog seq; & Type III RE are simpler, req no ATP & cleave DNA w/in the recog seq itself, ie.
Cuts at palindromes. Some RE makes stanggered cuts on 2 DNA strands leaving 2-4
nucleotides of 1 strand unpaired at each resulting end, these unpaired strands are called
sticky ends; they can base pair w/ each other or w/ other complementary sticky ends of other
DNA fragments. RE cleave both strands of DNA at the opposing phosphodiester bonds
leaving no unpaired bases on the end called blunt ends. A common intermediate step in
cloning of a specific gene/DNA segment is the construction of a DNA library (collection of
DNA clones)
-Cleavage of DNA molecules by RE: target DNA fragment is isolated & DNA ligase
can be used to join it to a similarly digested cloning vector, ie. Vector digested by the same
RE, eg. A fragment generated by EcoRI will not link to a fragment generated by BamHI. DNA
ligase catalyzes formation of new phosphodiester bonds in rxn that uses ATP or another
similar cofactor. The base pairing of complementary sticky ends greatly facilitates litigation
rxn but blunt ends less eff. Researchers can create new DNA seq by inserting synthetic DNA
fragments (called linkers) b.w ends that are being ligated. Polylinkers are inserted DNA
fragments w/ multiple recog seq for RE.
-Fig9-2: RE recognize & cleave only specific seq leaving sticky ends or blunt ends.
Fragments can be ligated to other DNAs, eg. Cleaved plasmid cloning vector. The rxn is
facilitated by annealing of complementary sticky ends; ligation is less eff for DNA fragments
w/ blunt ends than for those w/ complementary sticky ends & DNA fragments w/ diff sticky
ends generally are not ligated. A synthetic polylinker w/ recog seq for diff RE can be inserted

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into the plasmid that has been cleaved by a RE; the insert is a linker & insert w/ multiple
restriction sites is called a polylinker.
-Cloning vectors allow amplification of inserted DNA segments: 3 popular cloning vectors
commonly used w/ ecoli are plasmids, bacteriophages & bacterial artificial chromosomes & a
vector used to clone large DNA segments in yeast.
-Plasmids are circular DNA molec that replicate separately from the host
chromosome, eg. Constructed E.coli plasmid pBR322 (can clone 10-20 copies per cell); can
be introduced into bacterial cells via transformation—cells & plasmid DNA are incubated
together at 0° in CaCl2 soln then subjected to shock vs. electroporation—cells incubated w/
plasmid DNA are subjected to high-voltage pulse making bacterial membrane permeable to
large molec. The usual strategy is to use a plasmid that incl a gene that the host cell req for
growth under specific conditions, eg. Gene w/ resistance to an antibiotic; only cells
transformed by the recombinant plasmid can grow in the presence of antibiotic making any
cell that contains plasmid “selectable” under those growth conditions, such gene is called
selectable marker. Transformation of typical bacterial cells w/ purified DNA becomes even
more less successful as plasmid size inc & difficult to clone DNA segments longer than
15,000bp when plasmids are used as a vector.
*Basic DNA cloning: via transformation & electroporation in prokaryotes vs. transfection in
eukaryotes.
-Bacteriophages lamda has very eff mechanism for delivering its 48,502bp of DNA
into bacterium & can be used as a vector to clone some larger DNA segments upo to 23kbp.
Researches have developed bacteriophages lamda vectors that can be readily cleaved into
3 pcs, 2 contain essential genes (~30kbp) & 3rd pc is a filler DNA which is discarded when
the vector is to be used for cloning & additional DNA is inserted b/w 2 essential segments to
generate ligated DNA molecules long enough to form phage particles, ie. The packaging
mechanism selects for recombinant viral DNAs. Once the bacteriophage lamda fragments
are ligated to foreign DNA fragments of suitable size, the resulting recombinant DNAs can be
packaged into phage particles by adding them to crude bacterial extracts that contain all the
pro needed to assemble a complete phage, in what is known as in vitro packaging.
-Bacterial artificial chromosomes (BAC) are simply plasmid designed for cloning very
long segments (typically 100 to 300kbp of DNA); generally incl selectable markers such as
resistance to antibiotic chloramphenicol (Cmr) & very stable ori that maintains plasmid at 1 to
2 copies per cell. DNA fragments are cloned into the BAC vector, large circular DNAs are
introduced into host bacteria by electroporation which use host bacteria w/ mutations that
compromise the structure of their cell wall permitting uptake of the large DNA molec. Fig 9-6:
The par genes derived from the F plasmid assist in the even distribution of plasmids to
daughter cells at cell division which inc likelihood of each daughter cell carrying 1 copy of the
plasmid even when few copies are present. This low # of copies are useful in cloning large
segments of DNA b/c it limits opportunities for unwanted recombination rxns that can
unpredictably alter large cloned DNAs over time. The BAC incl selectable markers, eg. LacZ
gene is situated in the cloning region such that it is inactivated by cloned DNA inserts. Intro
of recombinant BACs into cells by electroporation is promoted by the use of cells w/
altered/porous cell wall. Recom DNAs are screened for Cmr; plates also contain a substrate
for beta-galactosidase that yields a coloured product. Colonies w/ active beta-galactosidase
& hence no DNA insert in the BAC vector turn blue & those w/o it, hence w/ desired DNA
inserts are white.
-Yeast artificial chromosomes (YACs): shuttle vectors are plasmids that can be
propagated in cells of 2/more diff species. YAC vectors are high capacity vectors that contain
all elements needed to maintain a eukaryotic chromosome in yeast nucleus (yeast ori, 2
selectable markers & specialized seq. Vector is propagated as a circular bacterial plasmid,
cleavage w/ BamHI removes a length of DNA b/w 2 telomere seq (TEL) leaving telomeres at
the ends of linearized DNA. Cleavage at another internal site, EcoRI divides the vector into 2
DNA segments referred to as vector arms, each w/ a diff selectable marker. Genomic DNA is
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prepared by partial digestion w/ RE to obtain suitable fragment size which are then
separated by pulsed field gel electrophoresis; the DNA fragments of appropriate size are
mixed w/ prepared vector arms & ligated; the mixture is then used to transform treated yeast
cells w/ very large molec (prepared by removal of cell wall to form spheroplast) which are
then selected for X/Y. The culture on the med that req presence of both selectable marker
genes ensure growth of only those yeast cells that contain YAC clone. Stability of YAC clone
inc w/ size; YACs that lack a telomere at either end are rapidly degraded.
-Specific DNA seq are detectable by hybridization: a probe is a radioactive labelled
DNA/RNA & complementary to DNA of interest. An agar plate w/ bacterial
coloniesNitrocellulose paper is pressed onto an agar plate containing many individual
colonies from the library, each w/ its own recombinant DNA; some cells from each colony
adhere to the paperpaper is treated w/ alkali to disrupt the cells & expose denatured
DNARadiolabelled DNA probe is incubated w/ paper & then washedadded radioactive
DNA probe anneals only to its complementary DNA & after unannealed probe is washed
away, the hybridized DNA can be detected by autoradiography.
-B/c more than 1 DNA seq can code for any given aa seq, the genetic code is said to
be degenerate thus the correct DNA seq for a known aa seq can’t be known in advance. The
probe is designed to be complementary to a region of the gene w/ minimal degeneracy, ie.
The region w/ the fewest possible codons for the aa (2 at most); oligos are synthesized w/
selectively randomized seq so that they contain either of the 2 possible nucleotides at each
posn of pot degeneracy, eg. Fig9-9; 1 of 8 will complement gene perfectly & all eight will
match at least 17 of 20 posns.
-Expression of cloned genes produces large quantities of pro: Most eukaryotic genes lack
the DNA seq elements, eg. Promoters, seq that instruct RNA polymerase where to bind, req
for expression their expression in E.coli cells so bacterial regulatory seq for transcription &
translation must be inserted at appropriate posn relative to the eukaryotic gene in the vector
DNA. Expression vectors are cloning vectors w/ transcription & translation signals needed for
the regulated expression of a cloned gene. The rate of expression of cloned gene is
controlled by replacing the gene’s promoter & regulatory seq w/ more eff & convenient
versions supplied by the vector. Fig 9-10: The gene to be expressed is inserted into 1 of the
restriction sites in the polylinker, near the promoter w/ the end encoding the amino end
proximal to the promoter. The promoter allows eff transcription of the inserted gene, the
transcription termination seq sometimes improves the amt & stability of mRNA produced.
The operator permits regulation by means of repressor that binds to it. The ribosome binding
site provides seq signals needed for eff translation of mRNA derived from the gene. The
selectable maker allows the selection of cells containing the recombinant DNA, eg. Antibiotic
resistance.
-Alterations in cloned genes produce modified pro: Specific aa may be replaced individually
by site-directed mutagenesis (SDM—by Michael Smith, won NP 1992) which changes aa
seq of pro by altering the DNA seq of cloned gene. 2 approaches: a) if appropriate restriction
sites flank the seq to be altered, researchers can simply cleave a DNA segment in a
recombinant DNA plasmid, replace it w/ a synthetic DNA fragment w/ specific-base pair
change & ligate it to the plasmid so now the plasmid contains the gene w/ the desire base
pair change. B) when suitably located restriction sites are not present, researchers can
perform oligonucleotide-directed mutagenesis where you can create a specific DNA seq
change. You have a single strand of recombinant plasmid DNA w/ specific DNA seq, CAG,
an oligonucleotide w/ the specific base change (GCC) is annealed to the specific DNA seq.
The annealed strand serves as a primer for synthesis of a strand complementary to the
plasmid vector; its fused via DNA polymerase, dNTPs (deoxynucleotide triphosphates) &
DNA ligase. Plasmid vector w/ annealed strand undergoes transformation of E.coli cells
repaired by cellular DNA enzymes. In E.coli cells, about ½ of plasmids will have gene w/
desired base-pair change. *Fusion pro is parts of 2 diff genes ligated to create new combos.
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