Download Chapter 4 Molecular Cloning Methods

Chapter 4
Molecular Cloning Methods
Introduction
The significance of gene cloning
To elucidate the structure and function of genes.
i.e.: investigating hGH gene
hGH gene: <10-6 of human genome
Problem 1: need kilograms of human
genome DNA for 1μg hGH gene
Problem 2: how to separate the gene from the
rest DNA
4.1 Gene Cloning
The procedure in a gene cloning experiment
is
1.
To place a foreign gene into a bacterial cell;
2.
To grow a clone of those modified bacteria.
The principle factors for gene cloning
experiment:

Restriction endonucleases

Vectors

Specific probe
 The Role of Restriction Endonucleases
 Vectors
Plasmids as Vectors
Phages as Vectors
λ Phage Vectors
Cosmids
M13 phage vectors
Phagemids
Eukaryotic Vectors
 Identifying a Specific Clone with a Specific Probe
Polynucleotide Probes
4.1.1 The Role of Restriction
Endonucleases



Restriction : restrict the host range of the
virus
Endonucleases : cut at sites within the
foreign DNA
How to name: the first 3 letters of the
Latin name of the microorganism + the
strain designation + Roman numeral
Recognition sequence
Recognition frequency
4 bp
44 =254 bp
6 bp
46=4096 bp
8 bp
48=65000bp
Rere cutters
The main advantage of restriction enzyme is there
ability to cut a DNA reproducibly in the same
place; this is the basis of many techniques used to
analyze genes.
Many restriction enzymes make staggered cut in the
two DNA strands, leaving a sticky ends, that can
base-pair together briefly.
Enzymes that recognize identical sequences are called
isoschizomers.
Restriction- modification system
R-M system
Almost all restriction nucleases are
paired with methylases that recognize
and methylate the same DNA sites
Figure 4.1 Maintaining
restriction endonuclease
resistance after DNA replication
We begin with an EcoRI site that is
methylated (red) on both strands. After
replication, the parental strand of each
daughter DNA duplex remains methylated,
but the newly made strand of each duplex
has not been methylated yet. The one
methylated strand in these hemimethylated
DNAs is enough to protect both strands
against cleavage by EcoRI. Soon, the
methylase recognizes the unmethylated
strand in each EcoRI site and methylates it,
regenerating the fully methylated DNA.
Figure 4.2 The first cloning experiment
involving a recombinant DNA assembled in
vitro.
Boyer and Cohen cut two plasmids, pSC101 and
RSF1010, with the same restriction endonuclease,
EcoRI. This gave the twolinear DNAs the same
stickyends, which were then linked in vitro using DNA
ligase. The investigators reintroduced the recombinant
DNA into E. coli cells by transformation and selected
clones that were resistant to both tetracycline and
streptomycin. These clones were therefore harboring
the recombinant plasmid.
Summary:
Restriction endonucleases recognize specific sequences in
DNA molecules and make cuts in both strands. This allows very
specific cutting of DNAs. Also, because the cuts in the two strands
are frequently staggered, restriction enzymes can create sticky ends
that help link together two DNAs to form a recombinant DNA in
vitro.
4.1.2 Vectors
Vectors serve as carriers to allow
replication of recombinant DNAs.

Origin of replication

Multiple cloning site(MCS)

Selection gene
Plasmids pBR322
Phages
λphage
Phagemids
pUC
cosmids
M13
Plasmids as Vectors
Summary:
The first generations of plasmid cloning vectors were pBR322 and
the pUC plasmids. The former has two antibiotic resistance genes and a
variety of unique restriction sites into which one can introduce foreign
DNA. Most of these sites interrupt one of the antibiotic resistance genes,
making screening straightforward. Screening is even easier with the
pUC plasmids. These have an ampicillin resistance gene and a multiple
cloning site that interrupts a partial β-galactosidase gene. One screens
for ampicillin-resistant clones that do not make active β-galactosidase
and therefore do not turn the indicator, X-gal, blue. The multiple
cloning site also makes it convenient to carry out directional cloning
into two different restriction sites.
Figure 4.3 The plasmid pBR322, showing the locations of 11 unique restriction
sites that can be used to insert foreign DNA
The locations of the two antibiotic resistance genes (Ampr =ampicillin resistance; Tetr
=tetracycline resistance) and the origin of replication (ori ) are also shown. Numbers
refer to kilobase pairs (kb) from the EcoRI site.
Figure 4.4 Cloning foreign DNA
using the PstI site of pBR322.
We cut both the plasmid and
the insert (yellow) with PstI, then
join them through these sticky
ends with DNA ligase. Next, we
transform bacteria with the
recombinant DNA and screen for
tetracycline-resistant, ampicillinsensitive cells. The recombinant
plasmid no longer confers
ampicillin resistance because the
foreign DNA interrupts that
resistance gene (blue).
Figure 4.5 Screening bacteria by
replica plating.
(a) The replica plating process. We
touch a velvet-covered circular tool to
the surface of the first dish containing
colonies of bacteria. Cells from each of
these colonies stick to the velvet and
can be transferred to the replica plate in
the same positions relative to each other.
(b) Screening for inserts in the pBR322
ampicillin resistance gene by replica
plating. The original plate contains
tetracycline, so all colonies containing
pBR322 will grow. The replica plate
contains ampicillin, so colonies bearing
pBR322 with inserts in the ampicillin
resistance gene will not grow (these
colonies are depicted by dotted circles).
The corresponding colonies from the
original plate can then be picked.
pUC
lacZ’ : coding for the amino
terminalportion of the enzyme β –
galactosidease.
Host E.coli strain carry a gene fragment
that codes the carboxyl potion of β –
galactosidease;
When X-gal cleaved by β –galactosidease, it
releases galactose plus an indigo dye that
stains the bacterial colony blue.
Figure 4.7 Joining of vector to insert. (a)
Mechanism of DNA ligase.
Step 1: DNA ligase reacts with an AMP
donor—either ATP or NAD(nicotinamide
adenine dinucleotide), depending on the
type of ligase. This produces an activated
enzyme (ligase-AMP). Step 2: The
activated enzyme donates a phosphate to the
free 5’-phosphate at the nick in the lower
strand of the DNA duplex, creating a highenergy diphosphate group on one side of the
nick. Step 3: With energy provided by
cleavage of the diphosphate, a new
phosphodiester bond is created, sealing the
nick in the DNA. This reaction can also
occur in both DNA strands at once, so two
independent DNAs can be joined together
by DNA ligase.
Figure 4.7 Joining of vector to insert.
(b)Alkaline phosphatase prevents vector
re-ligation.
Step 1: We cut the vector(blue, top left)
with BamHI. This produces sticky ends
with 5’-phosphates(red). Step 2: We
remove the phosphates with alkaline
phosphatase, making it impossible for the
vector to re-ligate with itself. Step 3: We
also cut the insert(yellow, upper right)
with BamHI, producing sticky ends with
phosphates that we do not remove. Step
4: Finally, we ligate the vector and insert
together. The phosphates on the insert
allow two phosphodiester bonds to
form(red), but leave two unformed bonds,
or nicks, These will be completed once
the DNA is in the transformed bacterial
cell.
Phages as vectors
Natural advantages over plasmid:
They infect cells much more efficiently
than plasmids transform cells, so the yield
of clones with phage vectors is usually
higher.
Summary:
Two kinds of phages have been especially popular as cloning vectors.
The first of these is λ, from which certain nonessential genes have been
removed to make room for inserts. Some of these engineered phages
can accommodate inserts up to 20 kb, which makes them useful for
building genomic libraries, in which it is important to have large pieces
of genomic DNA in each clone. Cosmids can accept even larger
inserts—up to 50 kb—making them a favorite choice for genomic
libraries. The second major class of phage vector is composed of the
M13 phages. These vector have the convenience of a multiple cloning
site and the further advantage of producing single-stranded recombinant
DNA, which can be used for DNA sequencing and for site-direct
mutagenesis. Plasmids called phagemids have also been engineered to
produce single-stranded DNA in the presence of helper phages.
Figure 4.8 Cloning in Charon 4.
(a) Forming the recombinant DNA.
We cut the vector (yellow) with EcoRI
to remove the stuffer fragment and save
the arms. Next, we ligate partially
digested insert DNA (red) to the arms.
(b) Packaging and cloning the
recombinant DNA. We mix the
recombinant DNA from (a) with an in
vitro packaging extract that contains λ
phage head and tail components and all
other factors needed to package the
recombinant DNA into functional phage
particles. Finally, we plate these
particles on E.coli and collect the
plaques that form.
Figure 4.9 Selection of positive
genomic clones by plaque
hybridization.
First, we touch a nitrocellulose ot
similar filter to the surface of the dish
containing the Charon 4 plaques from
Figure 4.8. Phage DNA released
naturally from each plaque will stick
to the filter. Next, we denature the
DNA with alkali and hybridize the
filter to a labeled probe for the gene
we are studying, then use X-ray film
to reveal the position of the label.
Cloned DNA from one plaque near
the center of the filter has hybridized,
as shown by the dark spot on the film.
Cosmids
Behave both as plasmids and as phages;
Contain the cos sites of λ and plasmid origin
of replication;
Have room for 40-50 kb inserts.
M13 phage vectors
β –galactosidease gene fragment
pUC family MCS
Single stranded DNA genome
Figure 4.10 Obtaining singlestranded DNA by cloning in M13
phage.
Foreign DNA (red), cut with HindIII, is
inserted into the HindIII site of the doublestranded phage DNA. The resulting
recombinant DNA is used to transform
E.coli cells, whereupon the DNA replicates
by a rolling circle mechanism, producing
many single-stranded product DNAs. The
product DNAs are called positive (+)
strands, by convention. The template DNA
is therefore the negative (-) strand.
Phagemides
Single-stranded;
Both phage and plasmid characteristics;
Help phage
Two RNA polymerase promoters (T7and T3)
Summary
Two kinds of phages have been especially popular as cloning vectors. The
first of these is λ, from which certain nonessential genes have been removed
to make room for inserts. Some of these engineered phages can accommodate
inserts up to 20 kb, which makes them useful for building genomic libraries,
in which it is important to have large pieces of genomic DNA in each clone.
Cosmids can accept even larger inserts—up to 50 kb—making them a favorite
choice for genomic libraries. The second major class of phage vector is
composed of the M13 phages. These vector have the convenience of a
multiple cloning site and the further advantage of producing single-stranded
recombinant DNA, which can be used for DNA sequencing and for site-direct
mutagenesis. Plasmids called phagemids have also been engineered to
produce single-stranded DNA in the presence of helper phages.
4.1.3 Identifying a Specific Clone with
a Specific Probe
Polynucleotide Probes
High stringency
Low stringency
Summary
Specific clones can be identified using
polynucleotide probes that bind to the gene
itself. Knowing the amino acid sequence of a
gene product, one can design a set of
oligonucleotides that encode part of this amino
acid sequence. This can be one of the quickest
and most accurate means of identifying a
particular clone.
4.2 The Polymerase Chain Reaction (PCR)
PCR amplifies a region of DNA
between two predetermined sites. Oligonucleotides complementary to these sites
serve as primers for synthesis of copies
of the DNA between the sites. Each cycle
of PCR double the number of copies of
the amplified DNA until a large quantity
has been made.
Figure 4.12 Amplifying DNA by the polymerase chain reaction.
Cycle 1: Start with a DNA duplex (top) and heat it to separate its two strands
(red and blue). Then add short, single-stranded DNA primers (purple and yellow)
complementary to sequences on either side of the region (X) to be amplified. The
primers hybridize to the appropriate sites on the separated DNA strands; now a
special heat-stable DNA polymerase uses these primers to start synthesis of
complementary DNA strands. The arrows represent newly made DNA, in which
replication has stopped at the tip of the arrowhead. At the end of cycle 1, two DNA
duplexes are present, including the region to be amplified, whereas we started with
only one. The 5’→3’ polarities of all DNA strands and primers are indicated
throughout cycle 1. The same principles apply in cycle 2. Cycle 2: Repeat the process,
heating to separate DNA strands, cooling to allow annealing with primers, and
letting the heat-stable DNA polymerase make more DNA. Now each of the four
DNA strands, including the two newly made ones, can serve as templates for
complementary DNA synthesis. The result is four DNA duplexes that have the
region to be amplified. Notice that each cycle doubles the number of molecules of
DNA because the products of each cycle join the parental molecules in serving as
templates for next cycle. This exponential increase yields 8 molecules in the next
cycle and 16 in the cycle after that. This process obviously leads to very high
numbers in only a short time.
4.2.1 cDNA Cloning
Nick translation
 Reverse transcriptase
 RNase H
 Terminal transferase

Figure 4.13 Making a cDNA library.
This figure focuses on cloning a single cDNA , but
the method can be applied to a mixture of mRNAs
and produce a library of corresponding cDNAs. (a)
Use oligo(dT) as a primer and reverse transcriptase
tocopy the mRNA (blue), producing a cDNA (red)
that is hybridized to the mRNA template. (b) Use
RNase H to partially digest the mRNA, yielding a
set of RNA primers base-paired to the first-strand
cDNA. (c) Use E.coli DNA polymerase I under nick
translation conditions to build second-strand
cDNAs on the RNA primers. (d) The second-strand
cDNA growing from the leftmost primer (blue) has
been extended all the way to the 3’-end of the
oligo(dA) corresponding to the oligo(dT) primer on
the first-strand cDNA. (e) To give the doublestranded cDNA sticky ends, add oligo(dC) with
terminal transferase. (f) Anneal the oligo(dC) ends
of the cDNA to complementary oligo(dG) ends of a
suitable vector (black). The recombinant DNA can
then be used to transform bacterial cells. Enzymes
in these cells remove remaining nicks and replace
any remaining RNA with DNA.
Figure 4.15 Using RT-PCR to clone a single
cDNA.
(a) Use a reverse primer (red) with a HindIII site
(yellow) at its 5’-end to start first-strand cDNA
synthesis, with reverse transcriptase to catalyze
the reaction. (b) Denature the mRNA-cDNA
hybrid and anneal a forward primer (red) with a
BamHI site (green) at its 5’-end. (c) This
forward primer initiates second-strand cDNA
synthesis, with DNA polymerase catalyzing the
reaction. (d) Continue PCR with the same two
primers to amplify the double-stranded cDNA.
(e) Cut the cDNA with BamHI and HindIII to
generate sticky ends. (f) Ligate the cDNA to the
BamHI and HindIII sites of a suitable vector
(purple). Finally, transform cells with the
recombinant cDNA to produce a clone.
Figure 4.16 RACE procedure to fill in the
5’-end of a cDNA.
(a) Hybridize an incomplete cDNA (red),
or an oligonucleotide segment of a cDNA
to mRNA (green), and use reverse
transcriptase to extend the cDNA to the
5’-end of the mRNA. (b) Use terminal
transferase and dCTP to add C residues
to the 3’end of the extended cDNA; also,
use RNase H to degrade the mRNA. (c)
Use an oligo(dG) primer and DNA
polymerase to synthesize a second
strand of cDNA (blue). (d) Perform PCR
with oligo(dG) as the forward primer
and an oligonucleotide that hybridizes
to the 3’-end of the cDNA as the reverse
primer. (e)The product is a cDNA that
has been extended to the 5’-end of the
mRNA. A similar procedure (3’-RACE)
can be used to extend the cDNA in the
3’-direction. In that case, there is no
need to tail the 3’-end of the cDNA with
terminal transferase because the mRNA
already contains poly(A); thus, the
reverse primer would be oligo(dT).
Summary
To make a cDNA library, we can synthesize cDNAs one strand at
a time, using mRNAs from a cell as templates for the first strands and
these first strands as temletes for the second strands. Reverse
trnscriptase generates the first strands and E.coli DNA polymerase I
generates the second strands. We can endow the double stranded
cDNAs with oligonucleotide tails that base-par with complementary
tails on a cloning vector. We can then use these recombinant DNAs to
transform bacteria. We can use RT-PCR to generate a cDNA from a
single type of mRNA, but we must know the sequence of the mRNA
in order to design the primers for the PCR step. If we put restriction
sites on the PCR primers, we place these sites at the ends of the
cDNA,so it is easy to ligate the cDNA into a vector. We can detect
particular clones by colony hybridazation with redioactive DNA
probes,or with antibodies if an expression vector such as λgt11 is used.
4.3 Methods of Expressing
Cloned Genes
4.3.1 Expression Vectors




Expression vectors with strong promoters
Inducible Expression Vectors
Expression vectors produce fusion proteins
Eukaryotic expression vectors
Figure 4.17
Figure 4.17 Producing a fusion
protein by cloning in a pUC plasmid.
Insert foreign DNA (yellow) into
the multiple cloning site (MCS);
transcription from the lac promoter
(purple) gives a hybrid mRNA
beginning with a few lacZ’ codons,
changing to insert sequence, then back
to lacZ’ (red). This mRNA is translated
to a fusion protein containing a few βgalactosidase amino acids for the
remainder ofthe protein. Because the
insert contains a translation stop codon,
the remaining lacZ’ codons are not
translated.
Figure 4.18 Using a PBAD vector.
The green fluorescent protein (GFP) gene was cloned into a vector under
control of the PBAD promoter and promoter activity was induced with increasing
concentrations of arabinose. GFP production was monitored by electrophoresing
extracts from cells induced with the arabinose concentrations given at top, blotting the
proteins to a membrane, and detecting GFP with an anti-GFP antibody .
Summary:
Expression vectors are designed to yield the protein
product of a cloned gene, usually in the greatest amount
possible. To optimize expression, these vectors provide
strong bacterial promoters and bacterial ribosome binding
sites that would be missing on cloned eukaryotic genes.
Most cloning vectors are inducible, to avoid premature
overproduction of a foreign product that could poison the
bacterial host cells.
Figure 4.19 Using an oligohistidine expression vector. (a) Map of a generic
oligohistidine vector.
Just after the ATG initiation codon (green) lies a coding region (red) encoding six
histidine in a row [(His)6]. This is followed by a region (orange) encoding a recognition
site for the proteolytic enzyme enterokinase (EK). Finally, the vector has a multiple
cloning site (MCS, blue). Usually, the vector comes in three forms with the MCS sites in
each of the three reading frames. One can select the vector that puts the gene in the
right reading frame relative to the oligohistidine.
Figure 4.19 Using an oligohistidine expression
vector. (b) Using the vector.
1. Insert the gene of interest (yellow) into the
vector in frame with the oligohistidine coding
region (red) and transform bacterial cells with the
recombinant vector. The cells produce the fusion
protein (red and yellow), along with other,
bacterial proteins (green).
2. Lyse the cells, releasing the mixture of proteins.
3. Pour the cell lysate through a nickel affinity
chromatography column, which binds the fusion
protein but not the other proteins.
4. Release the fusion protein from the column with
histidine or with imidazole, a histidine analogue,
which competes with the oligohistidine for binding
to the nickel.
5. Cleave the fusion protein with enterokinase.
6. Pass the cleaved protein through the nickel
column once more to separate the oligehistidine
from the desired protein.
Summary:
Expression vectors frequently produce fusion proteins,
with one part of the protein coming from coding
sequences in the vector and the other part from sequences
in the cloned gene itself. Many fusion proteins have the
great advantage of being simple to isolate by affinity
chromatography. The λgt11 vector produces fusion
proteins that can be detected in plaques with a specific
antiserum.
Figure 4.20 Forming a fusion protein
in λgt11.
The gene to be expressed (green) is
inserted into the EcoRI site near the end
of the lacZ coding region (red) just
upstream of the transcription terminator.
Thus, upon induction of the lacZ gene by
IPTG, a fused mRNA results, containing
the inserted coding region just
downstream of that of β-galactosidase.
This mRNA is translated by the host cell
to yield a fusion protein.
Figure 4.21 Detecting
positiveλgt11 clones by antibody
screening.
A filter is used to blot proteins
from phage plaques on a Petri
dish. One of the clones (red) has
produced a plaque containing a
fusion protein including βgalactosidase and a part of the
protein of interest. The filter with
its blotted proteins is incubated
with an antibody directed against
our protein of interest, then with
labeled Staphylococcus protein A,
which binds specifically to
antibodies. It will therefore bind
only to the antibody-antigen
complexes at the spot
corresponding to our positive
clone. A dark spot on the film
placed in contact with the filter
reveals the location of our
positive clone.
Figure 4.22 Expressing a gene in a baculovirus.
Figure 4.22 Expressing a gene in a baculovirus.
First, insert the gene to be expressed (red), into a baculovirus transfer vector.
In this case, the vector contains the powerful polyhedrin promoter (Polh), flanked
bythe DNA sequences (yellow) that normally surround the polyhedrin gene,
including a gene (green) that is essential for virus replication, the polyhedrin coding
region itself is missing from this transfer vector. Just downstream of the promoter is
a BamHI restriction site, which can be used to open up the vector (step a) so it can
accept the foreign gene (red) by ligation (step b). In step c, mix the recombinant
transfer vector with linear viral DNA that has been cut so as to remove the essential
gene. Transfect insect cells with the two DNAs together. This process is known as
co-transfection. The two DNAs are not drawn to scale, the viral DNA is actually
almost 15 times the size of the vector. Inside the cell, the two DNAs recombine by a
double crossover that inserts the gene to be expressed, along with the essential gene,
into the viral DNA. The result is a recombinant virus DNA that has the gene of
interest under the control of the polyhedrin promoter. Next, infect cells with the
recombinant virus. Finally, in step d and e, infect cells with the recombinant virus
and collect the protein product these cells make. Notice that the original viral DNA
is linear and it is missing the essential gene , so it cannot infect cells (f). This lack of
infectivity selects automatically for recombinant viruses; they are the only ones that
can infect cells.
Summary:
Foreign genes can be expressed in eukaryotic cells, and these
eukaryotic systems have some advantages over their prokaryotic
counterparts for producing eukaryotic proteins.
Two of the most important advantages are (1) Eukaryotic
proteins made in eukaryotic cells tend to be folded properly, so
they are soluble, rather than aggregated into insoluble inclusion
bodies. (2) Eukaryotic proteins made in eukaryotic cells are
modified (phosphorylated, glycosylated, etc.) in a eukaryotic
manner.
4.3.2 Other Eukaryotic Vectors


Yeast Artificial chromosomes (YACs)
Using the Ti plasmid to transfer genes to plants
Essential components of YAC vectors
•Centromers (CEN), telomeres (TEL) and
autonomous replicating sequence (ARS) for
proliferation in the host cell.
•ampr for selective amplification and markers such
as TRP1 and URA3 for identifying cells containing
the YAC vector.
•Recognition sites of restriction enzymes (e.g.,
EcoRI and BamHI)
BAC vectors
Bacterial artificial chromosomes are
based on the F factor of E. coli and can be
used to clone up to 350 kb of genomic DNA
in a conveniently handled E. coli host. They
are a morre stable and easier to use
alternative to YAC.
Using the Ti Plasmid to Transfer Gees to Plants
Nopaline and octopine Ti plasmids carry a variety
of genes, including T-regions that have overlapping
functions
T-DNA has almost identical repeats of 25 bp at each end in the
Ti plasmid. The right repeat is necessary for transfer and
integration to a plant genome. T-DNA that is integrated in a plant
genome has a precise junction that retains 1-2 bp of the right
repeat, but the left junction varies and may be up to 100 bp short
of the left repeat.
Figure 4.24 Crown gall tumors. (a) Formation of a crown gall.
1. Agrobacterium cells enter a wound in the plant, usually at the crown, or the junction of
root an stem. 2. The Agrobacterium contains a Ti plasmid in addition to the much larger
bacterial chromosome. The Ti plasmid has a segment (the T-DNA, red) that promotes tumor
formation in infected plants. 3. The bacterium contributes its Ti plasmid to the plant cell,
and the T-DNA from the Ti plasmid into grates into the plant’s chromosomal DNA. 4. The
genes in the T-DNA direct the formation of a crown gall, which nourishes the invading
bacteria.
Figure 4.24 Crown gall tumors. (b) Photograph of a crown gall tumor
genetated by cutting off the top of a tobacco plant and inoculating with
Agrobacterium.
This crown gall tumor is a teratoma, which generates normal as
well as tumorous tissues springing from the tumor.
Figure 4.25 Using a T-DNA plasmid to
introduce a gene into tobacco plant.
(a) A plasmid is formed with a foreign
gene (red) under the control of the mannopine
synthetase promoter (blue). This plasmid is
used to transform Agrobacterium cells.
(b) The transformed bacterial cells divide
repeatedly. (c) A disk of tobacco leaf tissue is
removed and incubated in nutrient medium,
along with the transformed Agrobacterium
cells. These cells infect the tobacco tissue,
transferring the plasmid bearing the cloned
foreign gene. (d) The disk of tobacco tissue
sends out roots into the surrounding medium.
(e) One of these roots is transplanted to
another kind of medium, where it forms a
shoot. This plantlet grows into a transgenic
tobacco plant that can be tested for expression
of the transplanted gene.
Summary:
Molecular biologists can clone hundreds of thousands
of base pairs of DNA at a time in yeast artificial
chromosomes (YACs). If they wish to transfer cloned
genes to plants, creating transgenic organisms with
altered characteristics, they use a plant vector such as the
Ti plasmid.