Download Cloning Vectors A cloning vector is a DNA molecule that can carry

Cloning Vectors
A cloning vector is a DNA molecule that can carry inserted DNA and be
perpetuated in a host system. It is also called cloning vehicle. There are many such
systems:
– Plasmids
– Cosmids
– Phages
– Phasmids
– Artificial vehicles: YAC, BAC etc.
Features of a ideal vector
1. Small size which is necessary for the efficiency of transfer of foreign DNA
2. Unique (single) restriction endonuclease recognition site into which the insert
DNA can be cloned
3. One or more selectable genetic markers for identifying recepient cells that carry
the cloning vector – insert DNA construct
Plasmids
It is an autonomous, double stranded, self-replicating, circular, extra
chromosomal DNA molecule. Virtually all bacterial genera have plasmids. Some
carry information for their own transfer from one cell to another – F plasmids. Some
encode resistance to antibiotics – R plasmid. Some carry specific set of genes for the
utilization of unusual metabolites – Degradative plasmids. And others have no
apparent function at all – Cryptic plasmids
Plasmids can range in size from less than 1 to more than 500 kb. Each plasmid
has a sequence that functions as the origin of replication of DNA – without this site it
cannot replicate in the host. A plasmid is considered to be a suitable cloning vector if
it possesses the following features:
1. Easily isolatable
2. Possessing single restriction site for one or more restriction enzymes
3. Insertion of linear molecule at on of theses sites should not alter its replication
properties
4. Reintroducible into host – but carrying identifiable marker – enabling easy
selection
5. Do not occur free in nature but are found in other bacterial cells
Plasmids used in genetic engineering are called vectors. Plasmids serve as important
tools in genetics and biotechnology labs, where they are commonly used to multiply
(make many copies of) or express particular genes. Many plasmids are commercially
available for such uses. The gene to be replicated is inserted into copies of a plasmid
containing genes that make cells resistant to particular antibiotics and a multiple cloning
site (MCS, or polylinker), which is a short region containing several commonly
used restriction sites allowing the easy insertion of DNA fragments at this location.
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Plasmid
Natural Occurrence
Size (Kb)
S. Marker
pACYC177
E. coli
3.7
Ampr, Kanr
pBR322
E. coli
4.0
Ampr, Tetr
pBR324
E. coli
8.3
Ampr ,Tetr
pMB9
R. coli
5.8
Tetr
pRK646
E. coli
3.4
Ampr
pC194
Staphylococcus aureus
3.6
Eryr
p SA 0501
S. aureus
4.2
Strr
p BS 161-1
Bacillus subtillus
3.65
Tetr
p WWO
Pseudomonas putida
117
Kanr
Next, the plasmids are inserted into bacteria by a process called transformation.
Then, the bacteria are exposed to the particular antibiotics. Only bacteria which take up
copies of the plasmid survive, since the plasmid makes them resistant. In particular, the
protecting genes are expressed (used to make a protein) and the expressed protein breaks
down the antibiotics. In this way the antibiotics act as a filter to select only the modified
bacteria.
Now these bacteria can be grown in large amounts, harvested and lysed (often using
the alkaline lysis method) to isolate the plasmid of interest. Another major use of
plasmids is to make large amounts of proteins. In this case, researchers grow bacteria
containing a plasmid harboring the gene of interest. Just as the bacteria produces proteins
to confer its antibiotic resistance, it can also be induced to produce large amounts of
proteins from the inserted gene. This is a cheap and easy way of mass-producing a gene
or the protein it then codes for, for example, insulin or even antibiotics.
However, a plasmid can only contain inserts of about 1–10 kbp. To clone longer
lengths of DNA, lambda phage with lysogeny genes deleted, cosmids, bacterial artificial
chromosomes or yeast artificial chromosomes could be used. One way of grouping
plasmids is by their ability to transfer to other bacteria. Conjugative plasmids contain socalled tra-genes, which perform the complex process of conjugation, the transfer of
plasmids to another bacterium. Non-conjugative plasmids are incapable of initiating
conjugation, hence they can only be transferred with the assistance of conjugative
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plasmids, by 'accident'. An intermediate class of plasmids are mobilizable, and carry only
a subset of the genes required for transfer. They can 'parasitize' a conjugative plasmid,
transferring at high frequency only in its presence. Plasmids are now being used to
manipulate DNA and may possibly be a tool for curing many diseases.
It is possible for plasmids of different types to coexist in a single cell. Several different
plasmids have been found in E. coli. But related plasmids are often incompatible, in the
sense that only one of them survives in the cell line, due to the regulation of vital plasmid
functions. Therefore, plasmids can be assigned into compatibility groups. Another way to
classify plasmids is by function. There are five main classes:
• Fertility-F-plasmids, which contain tra-genes. They are capable
of conjugation (transfer of genetic material between bacteria which are touching).
• Resistance-(R)plasmids, which contain genes that can build a resistance
against antibiotics or poisons and help bacteria producepili. Historically known as
R-factors, before the nature of plasmids was understood.
• Col-plasmids, which contain genes that code for (determine the production
of) bacteriocins, proteins that can kill other bacteria.
• Degradative plasmids, which enable the digestion of unusual substances,
e.g., toluene or salicylic acid.
• Virulence plasmids, which turn the bacterium into a pathogen (one that causes
disease).
Plasmids can belong to more than one of these functional groups. Plasmids that exist
only as one or a few copies in each bacterium are, upon cell division, in danger of being
lost in one of the segregating bacteria. Such single-copy plasmids have systems which
attempt to actively distribute a copy to both daughter cells. Some plasmids or microbial
hosts include an addiction system or "post segregational killing system (PSK)", such as
the hok/sok (host killing/suppressor of killing) system of plasmid R1 in Escherichia coli.
This variant produces both a long-lived poison and a short-lived antidote. Several
types of plasmid addiction systems (toxin/ antitoxin, metabolism-based, ORT systems)
were described in the literature and used in biotechnical (fermentation) or biomedical
(vaccine therapy) applications. Daughter cells that retain a copy of the plasmid survive,
while a daughter cell that fails to inherit the plasmid dies or suffers a reduced growth-rate
because of the lingering poison from the parent cell. Finally, the overall productivity
could be enhanced.
pBR322
pBR322 is a plasmid and for a time was one of the most commonly used E.
coli cloning vectors. Created in 1977, it was named eponymously after its Mexican
creators, p standing for plasmid, and BR for Bolivar and Rodriguez. pBR322 is 4361 base
pairs in length and contains a replicon region (source plasmid pMB1), the ampR gene,
encoding the ampicillin resistance protein (source plasmid RSF2124) and the tetR gene,
encoding the tetracycline resistance protein (source plasmid pSC101). The plasmid has
unique restriction sites for more than forty restriction enzymes. 11 of these 40 sites lie
within the tetR gene. There are 2 sites for restriction enzymes HindIII and ClaI within the
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promoter of the tetR gene. There are 6 key restriction sites inside the ampR gene. The
origin of replication or ori site in this plasmid is pMB1 (a close relative of ColE1). The
ori encodes two RNAs (RNAI and RNAII) and one protein (called Rom or Rop). The
circular sequence is numbered such that 0 is the middle of the unique EcoRI site and the
count increases through the tet genes. The ampicillin resistance gene is a penicillin betalactamase. Promoters P1 and P3 are for the beta-lactamase gene. P3 is the natural
promoter, and P1 is artificially created by the ligation of two different DNA fragments to
create pBR322. P2 is in the same region as P1, but it is on the opposite strand and
initiates transcription in the direction of the tetracycline resistance gene
pACYC177
•
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Type Cloning
Origin of Replicationp 15A
Copy #low, 15 copies per cell
Markers ampicillin kanamycin
Link to Sequence Compatible with ColE1
Cloning vectors – Phages
The bacteriophage lambda is important in molecular biology because it is used in
constructing vectors for gene cloning. Plasmids do this as well, but bacteriophages have
an advantage over plasmids. Plasmids are circular, indpendently replicating, doublestranded DNA, most often found in bacteria. They replicate quickly and are easily
manipulated in the laboratory. Plasmids are typically 2-10 thousand base pairs in size
(Corbley, 1999). While this small size allows plasmids the two aforementioned attributes,
it also means that plasmids are limited in the DNA fragments they can clone. They are
typically limited to fragments around 5 thousand base pairs (King, 2002). While plasmids
are great vector vehicles for many molecular endeavors, if the fragment to be cloned is
larger than the available insertion space of the plasmid, the plasmid vector will probably
not transfer the fragment of DNA successfully. Plasmid based cloning vectors can
generally carry only a 10 kb insert of DNA. For formation of a library it is helpful to have
larger fragments of DNA. Therefore a different vector is required. A bacteriophage is
considered a much more effective insertion vector for the formation of libraries. The most
commonly used phage vectors are derived form lambda (λ) phages that infect E. coli
A bacteriophage is any one of a number of viruses that infect bacteria.
Bacteriophages are among the most common biological entities on Earth. Typically,
bacteriophages consist of an outer protein capsid enclosing genetic material. The genetic
material can be ssRNA,dsRNA, ssDNA, or dsDNA along with either circular or linear
arrangement. Bacteriophages are much smaller than the bacteria they destroy. In its life
cycle the λ phage, infects E. coli, after injection of the viral DNA two possibilities exist.
It can enter a lytic cycle, which after 20 min results in the lysis of the host cell and the
release of about 100 phage particles. Alternatively the DNA can be integrated in the host
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genome as a prophage. The prophage can be maintained more or less indefinitely as a
benign guest [Lysogeny]. Under conditions of nutritional and environmental stress the
integrated prophage can be excised and enter the lytic cycle. The λ phage DNA is 50 kb
in length, of which approximately 20 kb are essential for the excision – integration
events. For forming genomic libraries this 20 kb is replaced by 20 kb of cloned DNA.
The resultant recombinant phage would go through compulsory lysogenic cycles
Lytic Cycle – Important Molecular events
The lytic cycle is typically considered the main method of viral replication, since
it results in the destruction of the infected cell. An infective phage has a head packed with
50 kb DNA. The production and assembly of the heads and tails and the packing of DNA
is considered to a highly coordinated sequence of events. The 50 kb in the head is a linear
molecule with a 12 base single strand extension at the 5prime end of each strand. These
extensions are called cohesive or cos ends. They contain sequences complementary to
each other. After the injection into the host the cos ends pair to form a circular DNA
molecule. During the early phase of the lytic cycle, replication from the circular molecule
generates a linear DNA strand that is a continuous length of many 50 kb molecules. Each
new head is filled with one 50 kb unit before the tail is attached
The volume of the head in an infective phage particle is 50 kb, if 38 kb is packed in the
head a non infective particle is produced. In contrast more than 52 kb can not fit into the
head capsule. The location of the cos sequence, which is 50 kb apart, ensures that each
head receives the correct amount of DNA. At the opening of the head is located a enzyme
that identifies the ds cos sequence and cuts the DNA. In experiments the phage DNA
was cut with BamHI, it produced 3 fragments
• The left arm region (L region) containing the genetic information for the
production of heads and tails
• The right arm region (R region) containing genes for replication and cell
lysis
• A middle fragment (I/E) that has the genes for the integration – excision
process
By this method it was determined that the middle fragment could be replaced by cloned
DNA. The length of the middle piece is around 20 kb
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Lysogenic cycle
The phage genome intergrates at an attachment (att) site with a partially
homologous on the E. coli genome. Two events are considered to be obligatory
to establish lysogenic cycle:
– (i) the synthesis of all late proteins must be stopped, and
– (ii) the lambda genome must integrate into the bacterial chromosome.
To prevent the synthesis of late proteins, the product of the cI gene must be synthesized.
The cI gene product is the lambda repressor protein. The latter, if synthesized, represses
the synthesis of all other lambda genome-encoded proteins. The cI gene occurs between
PL and PR (promoter left and promoter right) which are oriented in such a way that
neither transcribe the cl gene. The cI genes represses all the genes responsible for the
lytic pathways. No phage structural proteins are synthesized.The insertion of the proteins
of the cI genes (phage) and cro genes (E. coli) decides the events of the required
pathways either lytic or lysogenic. Only 50 % of the phage DNA is required for growth
and plaque formation
Advantages over plasmids
•
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•
DNA can be packed in vitro into phage heads and transduced into E. coli with
high efficiency
Foreign DNA up to 25 kb can be inserted into phage vector
Screening and storage of the rDNA is easier
Before using the phage as a vector it is necessary to remove the stuffer fragment
using restriction enzymes
Restriction sites can be obtained by inducung mutations or deletions
Two types of phage cloning vectors have been constructed
• Insertion vectors
• Replacement vectors
Insertion vector
• They have unique cleavage site into which relatively small pieces of DNA (35 –
53 kb) are inserted
• The maximum size of foreign DNA is about 18 kb
Replacement Vector
• They have cleavage sites present on either side of a length of non essential DNA
of phage
• As a result of cleavage left and right arms are formed each having a terminal cos
site
• The middle replaceable DNA is called stuffer DNA / stuffer region / stuffer
fragment
• The maximum size of insertable DNA depends on how much is non essential
• The substituted vectors are gt, WES, λ
• The non essential part can be separated from the arms by electrophoresis or
velocity gradient ultracentrifugation (size differences)
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•
•
•
•
Formation of multiple inserts can be used by using alkaline phosphatase before
ligation with insert fragment
r DNA formed by multiple inserts has too large a genome to be packed into the
head
Optimum distance between cos sites governs efficiency of packing
Bacteriophage libraries can be screened using DNA probes or immunological
assays
COSMIDS
A cosmid, first described by Collins and Hohn in 1978, is a type of
hybrid plasmid (often used as a cloning vector) that contains cos sequences, DNA
sequences originally from the Lambda phage. Cosmids can be used to build genomic
libraries.
Cosmids are able to contain 37 to 52 kb of DNA, while normal plasmids are able
to carry only 1–20 kb. They can replicate as plasmids if they have a suitable origin of
replication: for example SV40 ori in mammalian cells, ColE1 ori for double-stranded
DNA replication or f1 ori for single-stranded DNA replication in prokaryotes. They
frequently also contain a gene for selection such as antibiotic resistance, so that the
transfected cells can be identified by plating on a medium containing the antibiotic.
Those cells which did not take up the cosmid would be unable to grow.
Unlike plasmids, they can also be packaged in phage capsids, which allow the
foreign genes to be transferred into or between cells by transduction. Plasmids become
unstable after a certain amount of DNA has been inserted into them, because their
increased size is more conducive to recombination. To circumvent this, phage
transduction is used instead. This is made possible by the cohesive ends, also known
as cos sites. In this way, they are similar to using the lambda phage as a vector, but only
that all the lambda genes have been deleted with the exception of the cos sequence.
Cos sequences are ~200 base pairs long and essential for packaging. They contain
a cosN site where DNA is nicked at each strand, 12bp apart, by terminase. This causes
linearization of the circular cosmid with two "cohesive" or "sticky ends" of 12bp. (The
DNA must be linear to fit into a phage head.) The cosB site holds the terminase while it is
nicking and separating the strands. The cosQ site of next cosmid (as rolling circle
replication often results in linear concatemers) is held by the terminase after the previous
cosmid has been packaged, to prevent degradation by cellular DNases
Cosmids are predominantly plasmids with a bacterial oriV, an antibiotic selection
marker and a cloning site, but they carry one, or more recently two cos sites derived from
bacteriophage lambda. Depending on the particular aim of the experiment broad host
range cosmids, shuttle cosmids or 'mammalian' cosmids (linked to SV40 oriV and
mammalian selection markers) are available. The loading capacity of cosmids varies
depending on the size of the vector itself but usually lies around 40–45 kb. The cloning
procedure involves the generation of two vector arms which are then joined to the foreign
DNA. Selection against wildtype cosmid DNA is simply done via size exclusion.
Cosmids, however, always form colonies and not plaques. Also the clone density is much
lower with around 105 - 106 CFU per µg of ligated DNA.
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After the construction of recombinant lambda or cosmid libraries the total DNA is
transferred into an appropriate E.coli host via a technique called in vitro packaging. The
necessary packaging extracts are derived from E.coli cI857 lysogens (red- gam- Sam and
Dam (head assembly) and Eam (tail assembly) respectively). These extracts will
recognize and package the recombinant molecules in vitro, generating either mature
phage particles (lambda-based vectors) or recombinant plasmids contained in phage
shells (cosmids). These differences are reflected in the different infection frequencies
seen in favour of lambda-replacement vectors. This compensates for their slightly lower
loading capacity. Phage library are also stored and screened easier than cosmid
(colonies!) libraries.
Target DNA: the genomic DNA to be cloned has to be cut into the appropriate size
range of restriction fragments. This is usually done by partial restriction followed by
either size fractionation or dephosphorylation (using calf-intestine phosphatase) to avoid
chromosome scrambling, i.e. the ligation of physically unlinked fragments.
Based on the properties of DNA and Col E1 plasmids a group of Japanese scientists
(Fukumaki et al., 1976) showed that the presence of a small segment of λ phage DNA
containing cohesive end on the plasmid molecule is a sufficient prerequisite for in vitro
packaging of this DNA into infectious particles. A new breed of hybrid vectors was
thereby derived from the fusion of plasmids and bacteriophages. The first type were
called cosmids. Cosmids were developed by Collins and Hohn in 1978. They contain the
cos site of the bacteriophage DNA in association with the plasmid DNA. Cosmids lack
genes encoding for viral proteins – therefore neither viral particles are formed in the host
cell nor does lysis occur. Certain special features are observed in the cosmids
• The presence of the origin of replication
• A marker gene encoding for antibiotic resistance
• A special cleavage site for the insertion of foreign DNA
• Small size
• Presence of a cos site (12 bases)
The cos site helps to ligate and circularize the whole genome. The cosmids have a length
of 5 kb, the upper size limit of the foreign DNA fragment is approximately 45 kb.
According to the size of the cos sites and the upper size limit in the head of the phage, the
foreign DNA can be packed in vitro. Cosmids have been used as gene cloning vectors in
conjugation with the in vitro packaging system. The cosmid vector can be packed and the
resultant particle can be infected into a suitable host. The injected recombinant cosmid
DNA circularizes like phage DNA but replicated like a normal plasmid without the
expression of any phage functions. Transformer cells are selected on the basis of the
presence of a vector drug resistance marker. Cosmids provide an excellent means of
cloning large pieces of DNA. Because of their capacity for large fragments they are
particularly attractive vectors for the construction of libraries of eukaryotic genome
fragments. Partial digestion with restriction endonuclease provides suitably large
fragments. However there is a potential problem in the use of partial digests produced
this way. There is a possibility of two or more DNA fragments joining together in the
ligation reaction, this may create a clone containing fragments that were not initially
adjacent to each other in the genome. This would give an incorrect picture of their
genomic location. The problem can be over come by size fractionation of the partial
digest. Even with sized DNA, min practice cosmid clones may be produced that contain
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non-contiguous DNA fragments ligated from a single insert. This is solved by
dephosphorlyating the foreign DNA fragments so as to prevent their ligation together.
The above mentioned method is sensitive to the exact ratio of target to vector DNAs
because vector to vector ligation can occur. Furthermore recombinants with a duplicate
vector are unstable and breakdown in the host by recombination, resulting in the
propagation of non recombinant cosmid vectors. Such difficulties have been over come a
few examples are listed
• 1981 – Ish – Horowicz and Burke – pJB8 :
• purified left hand and right hand vector ends – incapable of self ligation
• Can accept dephosphorylated foreign DNA
• Eliminated the need to size the fragments and prevents the formation of
clones containing short foreign DNA or multiple vector sequences
• 1983 – Bates and Swift – c2XB :
• carries a BamH1 insertion site and two cos sites seperated by a blunt end
restriction site
• they ligate ineffectively under conditions used this too prevents self
ligation
• Modern cosmids of the pWE and sCos series contain the following features
• Multiple cloning sites for simple cloning using non size selected DNA
• Phage promoters flanking the cloning site
• Unique NotI, SacII or SfiI sites (rare cutters) flanking the cloning site to
permit the removal of the insert form the vector as a single fragment
Mammalian expression modules encoding dominant selectable markers may also be
present for gene transfer to mammalian cells if required. There are additional cosmid
vectors based on the λ phage as well as other phages that infect E. coli. The genome of P1
bacteriophage is 115 kb long and the cos mid can therefore carry an 85 kb insert. The
advantages of using a cosmid are twofold
• First the capacity of a cosmid is more than a plasmid,gene clusters and larger
genes are easier to clone
• Second, for creating a library, a large insert in the cloning vector means that fewer
clones have to be screened
pLFR-5
The commonly used cosmid pLFR-5 (approximately 6 kb) has
– two cos sites from the λ phage seperated by ScaI site
– A multiple cloning sequence with six unique sites (HindIII, PstI, SalI,
BamH1, SmaI and EcoRI)
– An origin of DNA replication (ori)
– Tetracycline resistant gene (Tet r)
This cosmid can carry around 40 kb DNA, that are purified by sucrose density gradient
centrifugation from a partial digestion of source DNA with BamHI. The pLFR-5 DNA is
initially cleaved by ScaI and the with BamHI. The two DNA samples are mixed and
ligated . Some of the products will have a 40 kb DNA piece inserted between the two
fragments that are derived from the digestion of the pLFR-5 DNA. These molecules will
be about 50 kb in length, with cos sequences 50 kb apart. These DNA constructs can be
successfully packed into a λ phage head in vitro. Since the packaging of the head accepts
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only 50 kb DNA reconstituted pLFR-5 without inserts will not be packed. After assembly
the phage particles the DNA is delivered by infection into E. coli.
Fosmids
Fosmids are similar to cosmids but are based on the bacterial F-plasmid. The
cloning vector is limited, as a host (usually E. coli) can only contain one fosmid
molecule. Fosmids are 40 kb of random genomic DNA. Fosmid library is prepared from a
genome of the target organism and cloned into a fosmid vector. Low copy number offers
higher stability than comparable high copy number cosmids. Fosmid system may be
useful for constructing stable libraries from complex genomes. Fosmid clones were used
to help assess the accuracy of the Public Human Genome Sequence. Fosmids are
plasmids that use the F-plasmid origin of replication and partitioning mechanisms to
allow cloning of large DNA fragments. A library that provides 20–70-fold redundant
coverage of the genome can easily be prepared
Bacterial artificial chromosome
A bacterial artificial chromosome (BAC) is a DNA construct, based on a
functional fertility plasmid (or F-plasmid), used for transforming and cloning in bacteria,
usually E. coli. F-plasmids play a crucial role because they contain partition genes that
promote the even distribution of plasmids after bacterial cell division. The bacterial
artificial chromosome's usual insert size is 150-350 kbp, but can be greater than 700 kbp.
A similar cloning vector called a PAC has also been produced from the bacterial P1plasmid. BACs are often used to sequence the genome of organisms in genome projects,
for example the Human Genome Project. A short piece of the organism's DNA is
amplified as an insert in BACs, and then sequenced. Finally, the sequenced parts are
rearranged in silico, resulting in the genomic sequence of the organism.
Common gene components
• oriS, repE - F
– for plasmid replication and regulation of copy number.
• parA and parB
– for partitioning F plasmid DNA to daughter cells during division and
ensures stable maintenance of the BAC.
• A selectable marker
– for antibiotic resistance; some BACs also have lac Z at the cloning site for
blue/white selection.
• T7 & Sp6
– phage promoters for transcription of inserted genes.
Contribution to models of disease
Inherited disease
BACs are now being utilized to a greater extent in modelling genetic diseases,
often alongside transgenic mice. BACs have been useful in this field as complex genes
may have several regulatory sequences upstream of the encoding sequence, including
various promoter sequences that will govern a gene's expression level. BACs have been
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used to some degree of success with mice when studying neurological diseases such as
Alzheimer's disease or as in the case of aneuploidy associated with Down syndrome.
There have also been instances when they have been used to study
specific oncogenes associated with cancers. They are transferred over to these genetic
disease models by electroporation/transformation, transfection with a suitable virus or
microinjection. BACs can also be utilised to detect genes or large sequences of interest
and then used to map them onto the human chromosome using BAC arrays. BACs are
preferred for these kind of genetic studies because they accommodate much larger
sequences without the risk of rearrangement, and are therefore more stable than other
types of cloning vectors.
Infectious disease
The genomes of several large DNA viruses and RNA viruses have been cloned as
BACs. These constructs are referred to as "infectious clones", as transfection of the BAC
construct into host cells is sufficient to initiate viral infection. The infectious property of
these BACs has made the study of many viruses such as
the herpesviruses, poxviruses and corona viruses more accessible. Molecular studies of
these viruses can now be achieved using genetic approaches to mutate the BAC while it
resides in bacteria.
PAC
The P1-derived artificial chromosome are DNA constructs that are derived from
the DNA of P1 bacteriophage. They can carry large amounts (about 100-300 kilobases)
of other sequences for a variety of bioengineering purposes. It is a type of vector used
to clone DNA fragments (100- to 300-kb insert size; average, 150 kb) in Escherichia
coli cells.
YAC
A yeast artificial chromosome (YAC) is a vector used to clone DNA fragments
larger than 100 kb and up to 3000 kb. YACs are useful for the physical mapping of
complex genomes and for the cloning of large genes. First described in 1983 by Murray
and Szostak, a YAC is an artificially constructed chromosome and contains
the telomeric, centromeric, and replication origin sequences needed for replication and
preservation in yeast cells. A YAC is built using an initial circular plasmid, which is
typically broken into two linear molecules using restriction enzymes; DNA ligase is then
used to ligate a sequence or gene of interest between the two linear molecules, forming a
single large linear piece of DNA. Yeast expression vectors, such as YACs, YIps (yeast
integrating plasmids), and YEps (yeast episomal plasmids), have an advantage over
bacterial artificial chromosomes (BACs) in that they can be used to express eukaryotic
proteins that require posttranslational modification. However, YACs have been found to
be less stable than BACs, producing chimeric effects.
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Human artificial chromosome
A human artificial chromosome (HAC) is a microchromosome that can act as a
new chromosome in a population of human cells. That is, instead of 46 chromosomes, the
cell could have 47 with the 47th being very small, roughly 6-10 megabases in size, and
able to carry new genes introduced by human researchers. Yeast artificial
chromosomes and bacterial artificial chromosomes were created before human artificial
chromosomes, which first appeared in 1997. They are useful in expression studies as
gene transfer vectors and are a tool for elucidating human chromosome function. Grown
in HT1080 cells, they are mitotically and cytogenetically stable for up to six months.
John J. Harrington, Gil Van Bokkelen, Robert W. Mays, Karen Gustashaw & Huntington
F. Willard of Case Western Reserve University School of Medicine published the first
report of human artificial chromosomes in 1997. They were first synthesized by
combining portions of alpha satellite DNA with telomeric DNA and genomic DNA into
linear microchromosomes
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