DNA Technology and Genomics

I. General outline of genetic engineering

A. DNA cleavage
B. Production of recombinant DNA
C. Cloning of the recombinant DNA
D. Screening clones
II. Manipulating DNA: DNA cleavage and Production of Recombinant DNA
A. restriction enzymes: molecular scissors with a twist
1. restriction enzymes, also called restriction endonucleases, are enzymes that cut DNA
molecules in specific places
2. restriction enzymes vary considerably
 hundreds of different kinds of restriction enzymes are known (recognizing different DNA
sequences)
 recognized sequence length varies (most common are “4-base cutters” and “6-base
cutters”)
 placement of cut varies; some leave “sticky ends”, others “blunt ends”
 most recognized sequences are palindromic
 the sequence on one strand matches that of the complementary strand read in the
opposite direction
 thus 5'-AGCGCT-3' would have a complementary strand 3'-TCGCGA-5' or reading
from 5' to 3', 5'-AGCGCT-3'
3. restriction enzymes are mostly from bacteria, and their natural role is to destroy DNA from
invading viruses
B. making recombinant DNA
1. restriction enzymes are used to cut up DNA of interest and a “vector” into which you want to
place the DNA, making restriction fragments
2. particularly when sticky ends are involved, the target DNA restriction fragment can form
basepairs with the vector
3. DNA ligase is then used to join the DNA strand backbones
III. Cloning of recombinant DNA: using vectors
A. cloning is the process of making many genetically identical cells from cell containing
recombinant DNA
1. the gene piece introduced in the recombinant DNA is said to be the DNA that is cloned
2. recombinant DNA is introduced to cells by a vector; the vector is usually maintained in the
altered cell line
B. a vector is a means of delivering recombinant DNA to an organism
1. vectors must have a way of getting into the host organism (transformation)
2. vectors must have some way of being propagated
 some types of vectors remain free but are copied and distributed in cell division
 some type of vectors have the inserted DNA integrate all or in part with the host DNA
3. vector DNA sequence must be known enough so that restriction sites can be accurately
predicted and used
C. most commonly, vectors are either plasmids, viruses, or yeast artificial chromosomes (YACs)
1. plasmids as vectors
 the most commonly used vectors today are plasmids
 plasmids are small, circular DNA molecules with at least one replication origin
 most bacterial cells contain several plasmids

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  some eukaryotic cells commonly have plasmids (such as the yeast Saccharomyces
cerevisiae)
 plasmids vary in what organisms can maintain them (largely based on the type of
replication origin they carry)
 most plasmids carry genes that are expressed, again with variations depending on the host
cell
2. viruses as vectors
 viruses infect cells with their DNA; recombinant DNA in a virus can thus be transferred
into cells
 some of this “transduction” occurs naturally, but genetic engineers control and exploit the
process
3. yeast artificial chromosomes (YACs) as vectors
 eukaryotes can support and maintain larger pieces of DNA as chromosomes
 YACs have the required elements of chromosomes (centromere, telomeres) and can be
used as vectors for large segments of recombinant DNA in some eukaryotes
D. vectors typically include a selectable marker and a cloning site
1. selectable markers usually are a gene for a product that the host cell cannot make, such as an
antibiotic resistance factor
2. the cloning site on a vector is engineered with many possible sites for restriction enzyme
cutting, where foreign DNA can be inserted
E. the piece of foreign DNA inserted at a cloning site is said to be cloned, and the combined
foreign DNA + vector is called recombinant DNA
IV. Screening
A. Often many clones are made with various DNA pieces inserted
B. Screening is used to find the DNA of interest; typically:
1. a selectable marker is used to ensure that the vector is present
2. a second type of selectable marker is tested to ensure that the vector contains inserted DNA
(that is, make sure it is recombinant DNA)
3. cells from cell colonies that pass the screens to this point are used as sources for making large
numbers of cells; DNA from these cells is then subjected to other treatments to help
identify cell lines containing the DNA of interest (for example, the probing covered later
in these notes)
V. DNA libraries
A. the first step in working with the DNA of a species is to break the whole genome into
manageable bits for study; this is done by creating DNA libraries
B. vectors serve as the “books” in a DNA library – each “book” has a different piece of inserted
DNA
C. two main types of libraries are genomic libraries and cDNA libraries
D. genomic libraries
1. raw genomic DNA is broken into fragments
 sometimes the breaking is done mechanically
 sometimes the breaking is done with restriction enzymes
 often a combination is used
2. the broken DNA pieces are put into vectors and then the vectors into host cells
3. cells lines are maintained for each library piece (often, the whole genome is represented
multiple times in the library for completeness)
4. the cell lines are given unique identifiers, and DNA probing techniques (described later) can
be used to determine what lines carry particular cloned DNA sequences
E. cDNA (complementary DNA) libraries

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 1. a more refined approach than genomic libraries, this type of library is based mainly on the
coding regions of DNA
2. mRNAs are isolated from a cell and converted into complementary DNA using the enzyme
reverse transcriptase
3. the cDNA is then inserted into vectors and the library is made and maintained just like a
genomic library
4. different types of cDNA libraries can be made, reflecting the conditions under which cells
made the original mRNAs
5. again, DNA probing techniques are used to find which lines have a cDNA of interest
VI. Techniques used to manipulate and study DNA before and after cloning include: PCR, DNA gel
electrophoresis, probing, DNA sequencing, and RFLPs
A. DNA sequence amplification: PCR (polymerase chain reaction) is used to get enough DNA to
work with
1. DNA polymerase can build a DNA strand provided there is a template strand, a primer, and
dNTPs (deoxyribonucleotides of each type: dATP, dCTP, dGTP, and dTTP)
 denaturation: heating a DNA molecule will eventually denature (“melt”) the double
strands into separate single strands, breaking the hydrogen bonds between A-T and C-
G basepairs; this can provide potential template strands
 annealing of primers: when the DNA cools, basepairs will reform; if small, specific
DNA primers are added in excess compared to the amount of target (template) DNA
molecules, the DNA primers will tend to bind to the target DNA strands and keep the
original double helices from reforming
 primer extension: then, DNA polymerases can add dNTPs to make a complementary
DNA strands, starting at the 3’ ends of the primers
 if you repeat this through a series of cycles, you will exponentially make new DNA
strands that cover a specific region of DNA, defined by the specific primers used – a
polymerase chain reaction, where each cycle essentially doubles the amount of your
target DNA fragment
2. PCR works well only when the DNA polymerase used can withstand temperatures that melt
DNA strands
 such enzymes are found in organisms that grow under very hot conditions (such as
thermophilic bacteria from hot springs)
 these are called heat-stable DNA polymerases (the best known one is Taq polymerase)
3. typically, the PCR works like this:
 DNA melting is done 94°C (just a bit under the boiling temperature of water) for a
minute or less
 DNA annealing is done at a temperature around 50°C for a minute or less, but this varies
depending on the DNA primers used and their optimal annealing temperature – you
want to avoid getting too cool, where some nonspecific annealing can occur
 DNA synthesis (primer extension) is performed at the optimal temperature for the heat-
stable DNA polymerase, usually 72°C; the time given to extension is roughly 1 min
per kilobase of DNA in the final target size
 the process then moves to back to DNA melting, and the “cycle” is repeated for up to ~35
cycles
 often, an extra melting period is put before any cycles (since larger DNA strands are
harder to melt), and an extra extension period is put after all cycles end (to finish up as
many strands as possible)
4. PCR greatly amplifies the target DNA sequence
 starting with a single double-stranded DNA molecule (the minimal extreme):
 after 4 cycles one has 24 = 16 DNA molecules

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  after 20 cycles one has 220 = 1,048,576 DNA molecules
 with wise primer choices, a segment of DNA can be amplified to make enough molecules
for useful study
 thus, given enough sequence knowledge and a small DNA sample, any piece of DNA
from any individual can usually be amplified through PCR to useful quantities
 PCR products can be used for such things as the raw material for cloning, for probing
DNA libraries, and for DNA sequencing
B. DNA gel electrophoresis
1. the overall DNA molecule is negatively charged, and will migrate though a viscous material
such as a gel if a voltage difference is supplied (moving toward the positive pole)
2. the speed of migration through a gel will be determined in part by the size of the DNA
molecule; the longer the molecule, the slower it moves
3. thus, relative migration rate through a gel can be used to determine the approximate size of a
DNA fragment
4. this mostly holds true for RNA as well, but different conditions must be used to prevent
degradation of RNA (which is much more common, and much more of a problem, than
DNA degradation)
C. probing
1. DNA and RNA fragments can be transferred to a filter, denatured, and incubated with probe
molecules that will hybridize (bond by forming correct basepairs) with specific sequences
2. the probe molecules (usually DNA fragments) can be made with some nucleotides that are
either radioactive or fluorescent, thus “labeling” the probe – and, when the probe is used,
labeling the sites on the filter where the probe is able to hybridize
3. if DNA is on the filter and being probed, this is called a Southern blot or DNA gel blot
(after the inventor)
4. if RNA is being probed, this is called a Northern blot or RNA gel blot
5. an analogous process with proteins is called a Western blot (there antibodies are used as
probes)
D. DNA sequencing
1. a DNA sequence can be determined using special nucleotides and migration differences of
DNA strands through a gel based on size
2. special ddNTPs (dideoxyribonucleotide triphosphates) are used for sequencing
3. when a ddNTP is incorporated into a growing DNA strand, it prevents further elongation of
the DNA strand
 there is no 3’-OH on which to add the next nucleotide, so the strand stops
 strand length is thus set by where the stop occurs
4. sequencing typically involves 4 polymerization mixtures
 usually, each mixture has labeled primers that allow DNA visualization
 each mixture also has a DNA polymerase and multiple copies of single-stranded template
DNA
 each mixture also has all 4 normal dNTPs
 the mixtures differ in the ddNTP included (one will be ddATP, one ddCTP, one ddGTP,
one ddTTP); the ddNTP is included in a small amount relative to the dNTPs
 at the end, each mixture will have a number of newly-synthesized DNA strands with a
variety of sizes, but within a given mixture you will know what ddNTP is at the 3’ end
of each of the stands
5. the labeled mixes are run on a gel and separated by size; the gel is then read from shortest
(thus fastest, and closest to the bottom) up to the longest (thus slowest, and closest to the
top) fragment – a letter is assigned based on the ddNTP that was used for the mixture that
was run in that gel lane

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 E. restriction fragment length polymorphisms (RFLPs)
1. DNA cut by restriction enzymes and run on a gel can produce distinguishable DNA bands
2. sequence differences between organisms (between species, or even within the same species)
can result in different bands
3. thus, given the right restriction enzymes or set of enzymes, RFLPs can serve as a “DNA
fingerprint” for an individual
4. DNA sequencing is the most reliable means of identification, and as it becomes cheaper and
more available it is replacing some uses of RFLP analysis; however, RFLP analysis likely
will always be quicker and cheaper than sequencing, and is still used heavily (RFLP
analysis is much more reliable than older techniques like actual fingerprint matching)
VII. Applications of genetic engineering (some examples)
A. Transgenic organism - any organism with a foreign gene(s) incorporated in it
B. Uses of transgenic organisms
1. as drug makers
 many gene products have potential medical uses; a useful protein or enzyme can often be
made in bulk via genetic engineering
 a case in point: the human insulin gene has been made in E. coli for over two decades
(much safer than insulin from other animals, which is not identical to human insulin)
 another example: human growth hormone – much safer than the old process of purifying
from cadavers
2. as medical models
 transgenic mice for modeling disease
 transgenic organisms for basic research on disease-related topics such as cell division
3. as gene therapy tools – viruses to deliver DNA to human cells
4. tools used to make better tools
 Cloned restriction enzymes, Taq polymerase, etc.
 sometimes cloned enzymes are modified to improve them for specific uses
5. improved food sources – examples:
 golden rice, engineered to make enough beta carotene to reduce vitamin A deficiencies in
many societies
 insect-resistant cotton, corn, etc. – express a bacterial gene that codes for a toxin that kills
insects that try to feed on the plant
 herbicide resistance
C. Safety guidelines
1. potential misuses (both intentional and accidental) are a concern
2. stringent guidelines are in place to prevent such things as producing and releasing a “super
virus,” “super bacteria,” or “super weed” that would become a serious medical and/or
ecological disaster
3. genetically engineered organisms that are released into the environment in some way are
closely monitored on a case-by-case basis
4. much concern over genetic engineering exists in the general public (especially in Europe), so
things such as labeling of genetically modified foods is a controversial issue

Southern Blotting

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Southern blotting was named after Edward M. Southern who developed this procedure
at Edinburgh University in the 1970s. To oversimplify, DNA molecules are
transferred from an agarose gel onto a membrane. Southern blotting is designed to
locate a particular sequence of DNA within a complex mixture. For example,
Southern Blotting could be used to locate a particular gene within an entire genome.

The amount of DNA needed for this technique is dependent on the size and specific
activity of the probe. Short probes tend to be more specific. Under optimal conditions,
you can expect to detect 0.1 pg of the DNA for which you are probing.

Procedure
1) DNA (genomic or other source) is digested with a restriction enzyme and separated
by gel electrophoresis, usually an agarose gel. Because there are so many different
restriction fragments on the gel, it usually appears as a smear rather than discrete
bands. The DNA is denature into single strands by incubation with NaOH.

2) The DNA is transfered to a membrane which is a sheet of special blotting paper.

The DNA fragementsw retain the same pattern of separation they had on the gel.

3) The blot is incubated with many copies of a probe which is single-stranded DNA.
This probe will form base pairs with its complementary DNA sequence and bind to
form a double-stranded DNA molecule. The probe cannot be seen but it is either
radioactive or has an enzyme bound to it (e.g. alkaline phosphatase or horseradish
peroxidase).

4) The location of the probe is revealed by incubating it with a colorless substrate that
the attached enzyme converts to a colored product that can be seen or gives off light
which will expose X-ray film. If the probe was labeled with radioactivity, it can
expose X-ray film directly.

Western Blotting

Background

Identification of a specific protein must be verified by more specific techniques, such as

immunoblotting, also called Western blotting.

Western Blot Definition: Western blotting is a technique used to identify and locate
proteins based on their ability to bind to specific antibodies.

Western Blotting is a method for identifying proteins that combines the resolving power
of PAGE with the specificity of antibodies. Because of the power of this technique,

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Western blotting has become an increasingly important tool, not only for protein
chemists but also in clinical labs where Western blotting is now considered the "gold
standard" for identifying HIV-positive individuals.

Western blotting is similar to Southern (DNA) and Northern (RNA) blotting in that target
molecules are first separated on a gel and then transferred from the gel to a
membrane, or blot, which binds the molecules. The membrane is then incubated with
reagents that specifically react with target molecules on the membrane. In Western
blotting, proteins are detected instead of nucleic acid and the reagent used to detect
proteins is an antibody, also a protein, instead of a nucleic acid probe. This antibody
specifically binds the target protein.

To understand the specificity of antigen-antibody reactions, the analogy is sometimes

made to the fit between a lock and key. While useful for visualization, this comparison
falls short. The lock and key fit is an interaction between two essentially flat surfaces
whereas an antigen antibody complex is more complicated; in part because it is a three
dimensional structure but also because of the electrostatic and hydrophobic interactions
between the amino acid residues of the two proteins. With this in mind, it is not
surprising that antibodies can be used to distinguish even very similar proteins and
even to identify other antibodies.

Western Blot Gives You Information on the:

Size of your Protein

Expression Amount of your Protein

Northern Blotting
Northern analysis despite its age in the high tech world of Real Time PCR, nuclease
protection assays (RPAs) and microarrays, is still the gold-standard for the detection
and quantitation of mRNA levels. This is because northern blot analysis allows a direct
comparison of the messenger RNA abundance between samples on a single membrane.

In northern blot the main difference between the other blotting techniques is that RNA
is the factor being detected. Also, due to the fact that RNA is usually single-stranded, it
creates complex secondary structures which affect its migration and hence denaturing
conditions are used to run the gels (unlike Southern).

RNA is separated out by RNA gel electrophoresis (usually agarose gel electrophoresis),
subsequent transfer to membrane, hybridization with probe, and finally detection.

Similarly to Southern blotting, the hybridization probes may be DNA or RNA in northern
blotting.

A variant of the procedure known as the reverse northern blot was occasionally
(although, infrequently) used. In this procedure, the substrate nucleic acid (that is
affixed to the membrane) is a collection of isolated DNA fragments, and the probe is
RNA extracted from a tissue and radioactively labelled.

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The use of DNA microarrays that have come into widespread use in the late 1990s and
early 2000s is more akin to the reverse procedure, in that they involve the use of
isolated DNA fragments affixed to a substrate, and hybridization with a probe made
from cellular RNA. Thus the reverse procedure, though originally uncommon, enabled
the one-at-a-time study of gene expression using northern analysis to evolve into gene
expression profiling, in which many (possibly all) of the genes in an organism may have
their expression monitored

Applications of the Northern Blot

Northern blots have been superseded in most areas by Real Time PCR and microarray
approaches. It is not often used for clinical or diagnostic purposes.

The northern blot protocol and its variations are used however in molecular biology
research to:

 A gold-standard for the direct study of gene expression at the level of mRNA
(messenger RNA transcripts).
 detection of mRNA transcript size
 study RNA degradation
 study RNA splicing - can detect alternatively spliced transcripts
 study RNA half-life
 often used to confirm and check transgenic / knockout mice (animals)

Restriction Enzymes
Restriction enzymes, also known as restriction endonucleases, are enzymes that cut a
DNA molecule at a particular place. They are essential tools for recombinant DNA
technology. The enzyme "scans" a DNA molecule, looking for a particular sequence,
usually of four to six nucleotides. Once it finds this recognition sequence, it stops and
cuts the strands. This is known as enzyme digestion. On double stranded DNA the
recognition sequence is on both strands, but runs in opposite directions. This allows the
enzyme to cut both strands. Sometimes the cut is blunt, sometimes the cut is uneven
with dangling nucleotides on one of the two strands. This uneven cut is known as sticky
ends.
A blunt end may look like this:

A sticky end like this:

Most plasmids used for recombinant technology have recognition sequences for a
number of restriction enzymes. This allows a scientist to choose from a number of

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places to cut the plasmid with a restriction enzyme. Ligation enzymes can then be used
to sort of paste in new genomic sequences. These mutated, or recombined, plasmids
can then be grown up in bacterial cells and used for a number of purposes, including
the addition of genes to mammalian genomes.

Protein purification protocol

PCR (polymerase chain reaction)

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Let's say you have a biological sample with trace amounts of DNA in it. You want to
work with the DNA, perhaps characterize it by sequencing, but there isn't much to work
with. This is where PCR comes in. PCR is the amplification of a small amount of DNA
into a larger amount. It is quick, easy, and automated. Larger amounts of DNA mean
more accurate and reliable results for your later techniques.
PCR can be used to create a DNA "fingerprint," which is unique to each individual.
These DNA fingerprints can be useful in real-world applications relating to
paternity/maternity, kinship, and forensic testing.
The technique was developed by Nobel laureate biochemist Kary Mullis in 1984 and is
based on the discovery of the biological activity at high temperatures of DNA
polymerases found in thermophiles (bacteria that live in hot springs).
Most DNA polymerases (enzymes that make new DNA) work only at low temperatures.
But at low temperatures, DNA is tightly coiled, so the polymerases don't stand much of
a chance of getting at most parts of the molecules.
But these thermophile DNA polymerases work at 100C, a temperature at which DNA is
denatured (in linear form). This thermophilic DNA polymerase is called Taq polymerase,
named after Thermus aquaticus, the bacteria it is derived from.
Taq polymerase, however, has no proofreading ability. Other thermally stable
polymerases, such as Vent and Pfu, have been discovered to both work for PCR and to
proofread.

You'll need four things to perform PCR on a sample:

1. The target sample. This is the biological sample you want to amplify DNA from.

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2. A primer. Short strands of DNA that adhere to the target segment. They identify the
portion of DNA to be multiplied and provide a starting place for replication.
3. Taq polymerase. This is the enzyme that is in charge of replicating DNA. This is the
polymerase part of the name polymerase chain reaction.
4. Nucleotides. You'll need to add nucleotides (dNTPs) so the DNA polymerase has
building blocks to work with.
There are three major steps to PCR and they are repeated over and over again, usually
25 to 75 times. This is where the automation is most appreciated.
1. Your target sample is heated. This denatures the DNA, unwinding it and breaking the
bonds that hold together the two strands of the DNA molecule, leaving you with single
stranded DNA (ssDNA).
2. Temperature is reduced and the primer is added. The primer molecules now have the
opportunity to bind (anneal) to the pieces of ssDNA. This labels the portions of DNA to
be amplified and provides a starting place for replication.
3. New pieces of ssDNA are made. Taq polymerase catalyzes the generation of new
pieces of ssDNA that are complimentary to the portions marked by the primers. The job
of Taq polymerase is to move along the strand of DNA and use it as a template for
assembling a new stand that is complimentary to the template. This is the chain
reaction in the name polymerase chain reaction.
PCR is so efficient because it multiplies the DNA exponentially for each of the 25 to 75
cycles. A cycle takes only a minute or so and each new segment of DNA that is made
can serve as a template for new ones.
Perhaps the most important thing to remember is to be very aware of contamination.
If, for example, you unknowingly slough off a piece of skin into your sample, then your
DNA may be amplified in the PCR reaction.
Here are some other factors to optimize your results with PCR:
1. Annealing temperature. Starts at the low end of what you think will work, then move
up as necessary. If the temperature is too low, the primers will make more mistakes
and you'll get too many bands when you run your sample on a gel. If the temperature
is too high you will get no results and your gel will be blank. You want to be about 3C to
5C below the melting temperature (Tm). A rough formula for determining Tm is
Tm=4(G + C) + 2(A + T).
2. Magnesium concentration. You want your Mg2+ concentration to be about 1.5mM to
3mM. If you go too high, the polymerase will make more mistakes.
3. Think carefully about primer design. Both primers should have approximately the
same Tm so they both anneal at the same temperature. Two out of three bases on the

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3' end should b G or C to get good hybridization (G and C have three H-bonds so you
get better polymerization). Lastly, avoid primer dimers, which occur when the primers
have ends that will anneal to each other. This will produce NO product.
4. More is not necessarily better. More polymerase produces more nonspecific product,
so don't just carelessly dump in a bunch of polymerase. Additionally, PCR reactions
don't work if there is too much DNA.
RT-PCR
Taq polymerase does not work on RNA samples, so PCR cannot be used to directly
amplify RNA molecules. The incorporation of the enzyme reverse transcriptase (RT),
however, can be combined with traditional PCR to allow for the amplification of RNA
molecules. After you add your RNA sample to the PCR machine, add a DNA primer as
usual and allow it to anneal to your target molecule. Then add RT along with dNTPs,
which will elongate the DNA primer and make a cDNA copy of the RNA molecules and
run the PRC reaction as usual. The product of RT-PCR is a double stranded DNA
molecule analogous to the target segment of the RNA molecule.

Plasmids
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A plasmid is an independent, circular, self-replicating DNA molecule that carries only a
few genes. The number of plasmids in a cell generally remains constant from generation
to generation. Plasmids are autonomous molecules and exist in cells as
extrachromosomal genomes, although some plasmids can be inserted into a bacterial
chromosome, where they become a permanent part of the bacterial genome. It is here
that they provide great functionality in molecular science.
Plasmids are easy to manipulate and isolate using bacteria (see also alkaline lysis) They
can be integrated into mammalian genomes, thereby conferring to mammalian cells
whatever genetic functionality they carry. Thus, this gives you the ability to introduce
genes into a given organism by using bacteria to amplify the hybrid genes that are
created in vitro. This tiny but mighty plasmid molecule is the basis of recombinant DNA
technology.
There are two categories of plasmids. Stringent plasmids replicate only when the
chromosome replicates. This is good if you are working with a protein that is lethal to
the cell. Relaxed plasmids replicate on their own. This gives you a higher ratio of
plasmids to chromosome.
So how do we manipulate these plasmids?
1. Mutate them using restriction enzymes, ligation enzymes, and PCR. Mutagenesis is
easily accomplished by using restriction enzymes to cut out portions of one genome and
insert them into a plasmid. PCR can also be used to facilitate mutagenesis. Plasmids are
mapped out indicating the locations of their origins of replication and restriction enzyme
sites.
2. Select them using genetic markers. Some bacteria are antibiotic resistant. While this
is a serious health problem, it is a godsend to molecular scientists. The gene that
confers antibiotic resistance can be added (ligated) to the gene you are inserting into
the plasmid. So every plasmid that contains your target gene will not be killed by
antibiotics. After you transfect your bacterial cells with your engineered plasmid (the
one with the target gene and the antibiotic resistant marker), you incubate them in a

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nutrient broth that also contains antibiotic (usually ampecillin). Any cells that were not
transfected (this means they do not have your target gene in them) are killed by the
antibiotic. The ones that do have the gene also have the antibiotic resistant gene, and
therefore survive the selection process.
3. Isolate them (such as with alkaline lysis)
4. Transform them into cells where they become vectors to transport foreign genes into
a recipient organism.
There are some minimum requirements for plasmids that are useful for recombination
techniques:
1. Origin of replication (ORI). They must be able to replicate themselves or they are of
no practical use as a vector.
2. Selectable marker. They must have a marker so you can select for cells that have
your plasmids.
3. Restriction enzyme sites in non-essential regions. You don't want to be cutting your
plasmid in necessary regions such as the ORI.
In addition to these necessary requirements, there are some factors that make
plasmids either more useful or easier to work with.
1. Small. If they are small, they are easier to isolate (you get more), handle (less
shearing), and transform.
2. Multiple restriction enzyme sites. More sites give you greater flexibility in cloning,
perhaps even allowing for directional cloning.
3. Multiple ORIs. It is important to note that two genes must have different ORIs if they
are going to be inserted in the same plasmid.

Sequencing
One of the major methods of DNA sequencing is known as chain termination
sequencing, or Sanger sequencing after its inventor, biochemist Frederick Sanger.

Sanger method

The method is elegantly simple.

While DNA chains are normally made up of deoxy-nucleotides (dNTPs), the Sanger
method uses di-deoxy-nucleotides.

Dideoxynucleotides (ddNTPs) are missing a hydroxy (OH) group at the 3' position. This
position is normally where one nucleotide attaches to another to form a chain. If there
is no OH group in the 3' position, the additional nucleotides cannot be added to the
chain, thus interrupting chain elongation.

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Manual sequencing

1. Begin the process by synthesizing a chain that is complimentary to the template you
want to analyze.
2. Add a specific primer that you know will anneal.
3. Divide your sample among four test tubes. One test tube will be used for each
specific nucleotide (dGTP, dATP, dCTP, and dTTP).
4. Add DNA polymerase to each tube and one specific nucleotide per tube.
5. Add ddNTPs to all four tubes. Once you add the ddNTPs, there is no way for the
chain to keep elongating, hence you have dd chain termination.
6. Run this on a polyacrylamide gel using one lane per reaction tube (dGTP, dATP,
dCTP, and dTTP).
7. To sequence, read the order of bases from the smallest to the largest.

Automated sequencing

Or, if this looks like too much work, you can pay for automated sequencing, where a
machine does most of the work for you. Of course, you'll need about $100,000 for the
machine.

An automated sequencer runs on the same principle as the Sanger method

(dideoxynucleotide chain termination). A laser constantly scans the bottom of the gel,
detecting bands as they move down the gel. Where the manual method uses
radioactive labeling, automated sequencing uses fluorescent tags on the ddNTPs (a
different dye for each nucleotide). This makes it possible for all four reactions (dGTP,
dATP, dCTP, and dTTP) to be run in one lane, so you can have huge numbers of
reactions on one gel. This is a very efficient method.

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