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•On September 14, 1990, researchers at the U.S. National Institutes of Health (NIH)
performed the first approved gene therapy procedure on a four-year-old girl
named Ashanti DeSilva.
•Born with a rare genetic disease, severe combined immune deficiency (SCID),
Ashanti lacked a healthy immune system and was extremely vulnerable to
infection.
DNA Technology
•Children with SCID usually develop overwhelming infections and rarely survive
to adulthood; even a common childhood illness like chicken pox is
life-threatening.
•Ashanti led a cloistered existence, avoiding contact with people outside her
family, remaining in the sterile environment of her home, and battling frequent
illnesses with massive amounts of antibiotics.
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•In Ashanti's gene therapy procedure, her own white blood cells were genetically
modified and then infused back into her bloodstream [introducing properly
functioning genes into as many of the cells as possible. The normal genes were
delivered through the use of a specially-engineered virus].
•Laboratory tests show that the therapy has strengthened Ashanti's immune
system. .
DNA Technology
•She no longer has recurrent colds, has been allowed to attend school, and has
been immunized against whooping cough.
•This gene therapy procedure is not a cure, however.
•The genetically-treated white blood cells only survive for a few months and
must then be replaced, but Ashanti's future is much brighter because of the new
therapy (VI, Thompson [The First] 1993).
DNA Technology
•Recombinant DNA technology refers to the set of techniques for recombining
genes from different sources in vitro and transferring this recombinant DNA into
a cell where it may be expressed.
DNA Technology
•Although the technique was available to grow plant and animal cells in culture,
the workings of their genomes could not be examined using existing methods.
•Recombinant DNA technology now makes it possible for scientists to examine the
structure and function of the eukaryotic genome, because it contains several key
components:
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•· Biochemical tools that allow construction of recombinant DNA
•· Methods for purifying DNA molecules and proteins of interest
•· Vectors for carrying recombinant DNA into cells and replicating it
•Techniques for determining nucleotide sequences of DNA molecules.
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•Restriction enzymes are major tools in recombinant DNA technology.
•First discovered in the late 1960s, these enzymes occur naturally in bacteria
where they protect the bacterium against intruding DNA from other organisms.
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•· This protection involves
restriction, a process in which the foreign DNA is cut into small segments.
•Most restriction enzymes -only recognize short, specific nucleotide sequences
called recognition sequences or restriction sites.
•They only cut at specific points within those sequences.
DNA Technology
•Restriction-Modification
•In the 1950's, evidence was found of a sort of primitive immune system in
bacteria. The bacteria cell seemed to be able to restrict the growth and
replication phages (bacteriophage-invading virus for that particular bacteria).
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•It was shown that the bacteria possessed an enzyme system that selectively
recognizes and destroys foreign phage DNA within the bacterial membrane and that
it also modifies the chromosomal DNA of the bacterium to prevent self
destruction.
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•These enzymes were eventually extracted and were known as restriction
endonucleases, enzymes that attack and digest internal regions of the DNA of the
invading bacteriophage but not that of the host.
•So these enzymes had a cutting/ restriction activity and a modification
(protecting) activity.
DNA Technology
•The protecting or modifying enzyme protests the host DNA from digestion by
adding methyl groups to the nucleotide within the sequence recognized by the
restriction enzyme.
•This then blocks the restriction enzyme from digesting the host DNA.
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•Because the 1st enzymes isolated were not precise in where they cut, they were
of no use to the scientists who first isolated them.
•However in 1970, an enzyme named HindII.
•First the restriction activity was separate from the modification activity and
it cleaves DNA predictably, cutting within its recognition sequence.
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•The occurrence of unique restriction sites allows the DNA molecule to be cut at
a single location by a restriction enzyme and that this could be mixed with
other cut DNA's to form a circular recombinant DNA molecule (termed plasmid).
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•DNA polymerase then fills in any ingle stranded gaps and DNA ligase, seals the
junction points between the new segments.
•Further down the line, this new plasmid is allowed to replicate within the host
bacteria cell which passes it on with each replication.
DNA Technology
•Restriction endonucleoases, or restriction enzymes are used as molecular
scalpels to cut DNA in a precise and predictable manner. They can break the
phosphodiester bonds that link adjacent nucleotides in DNA and RNA molecules.
DNA Technology
•There are 3 Classes of restriction endonucleases.
•Type I and type III enzymes have both restriction (cutting) and modification (methylating)
activity.
•Both types cut at sites some distance from their recognition sequences and ATP
is required to provide energy for movement of the enzyme along the DNA molecule
from recognition site to cleavage site.
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•Type I enzymes cut at random sites 1000 nucleotides or more away from the
recognition sequence while type III cut at specific sites quite near the
sequence but are difficult to predict.
•Those used in DNA science are type II.
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•Each have only a restriction activity...modification is carried out by another
enzyme,
•each cuts in a predicable and consistent manner at a site within or adjacent to
the recognition sequence and
•they only require magnesium ion as a cofactor not ATP.
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•Today, more than 3000 type II enzymes have been isolated from a variety of
prokaryotic organisms.
•
•By some unknown mechanism, type II restriction endonuclease scans a DNA
molecule, stopping only when it recognizes a specific sequence of nucleotides
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•Most recognize a four-or six nucleotide sequence.
•Assuming that the four component nuleotides (ATGC) are distributed randomly
within a DNA molecule, than any four nucleotides will occur on average every 256
nucleotides (4x4x4x4) and six sequence every 4096 nucleotides (4x4x4x4x4x4).
DNA Technology
•Most restriction enzymes have recognition sites that are composed of
symmetrical nucleotide sequences.
•This means the recognition sequence read forward on one DNA strand is identical
to the sequence read in the opposite direction on the complementary strand.
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•Also for each type II endonulease, there is a corresponding modifying enzyme
that blocks restriction activity by methylating specific nucleotides within the
recognition sequence.
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•Some endonuleases cut cleanly through the DNA helix by cleaving both
complementary strands at the same nucleotide position (usually at the center of
the site).
•These enzymes leave flush or blunt ended fragments.
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•Other endonuleases cleave each strand off center in the recognition site at
positions two to four nucleotides apart.
• This creates fragments it exposed ends of short, single stranded sequences.
•These single stranded overhangs are also called sticky ends and are useful in
making recombinant DNA molecules.
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•The exposed nucleotides serve as a template for realignment. A given
restriction enzyme cuts all DNA in exactly the same fashion, regardless of
whether the source is bacterium, plant, or human being.
•Thus the sticky ended fragment can be recombined with any other fragment
generated by the same restriction enzyme
DNA Technology
•Ultimately, the propagation of a DNA sequence must take place inside a living
cell. Thus, transformation, the cellular uptake and expression of DNA in a
bacterium is crucial to research process. Needs are 1. suitable host organism to
insert the gene, 2. self replicating vector to carry the gene into the host
organism and 3. a means of selecting the host cells that have taken up the gene.
DNA Technology
•The bacterium E. coli has become the most widely used organism. it has a single
chromosome of about 5 million base pairs (bpu).
•Because genetic code is universal, it can accept foreign DNA derived from any
organism. A foreign gene in E. coli is replicated and in some cases translated
in exactly the same manner as the native bacterial DNA.
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•E coli sees foreign DNA as its own.
•Even under the best circumstances, the uptake of a specific foreign gene is
rare and is most easily accomplished in a large population of organisms that are
reproducing rapidly.
•E. coli, under favorable conditions, can replicate once every 22 minutes give
rise to 30 generations with more than 1 billion cells in 11 hours.
DNA Technology
•Plasmid vector
•A vector is an organism that carries a pathogen from one host organism to
another.
•In molecular biology, a vector is a DNA molecule that is used to carry foreign
DNA sequences into E. coli or another host cell.
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•Plasmids are the simplest bacterial vectors.
•Ranging in length from 1000-200000 base pairs, they are circular DNA molecules
that exist separate from the main bacterial chromosome.
•It must contain specific DNA sequences to allow it to replicate within the host
cell.
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•Two types occur, stringent, exist as a single or a few copies and only
replicate with the main bacterial chromosome and relaxed which replicate
autonomously of the main chromosome and may have 10-500 per cell.
•Generally only plasmids that confer some select advantage are maintained in a
given bacterial population. (antibiotic resistance).
DNA Technology
•Polymerase chain reaction (PCR), a technique used to make numerous copies of a
specific segment of DNA quickly and accurately. The polymerase chain reaction
enables investigators to obtain the large quantities of DNA that are required
for various experiments and procedures in molecular biology, forensic analysis,
evolutionary biology, and medical diagnostics.
DNA Technology
•The machine designed to carry out PCR reactions can complete many rounds of
replication, producing billions of copies of a DNA fragment, in only a few
hours.
•The PCR technique is based on the natural processes a cell uses to replicate a
new DNA strand.
DNA Technology
•Only a few biological ingredients are needed for PCR. The integral component is
the template DNA--i.e., the DNA that contains the region to be copied, such as a
gene.
•As little as one DNA molecule can serve as a template.
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•The only information needed for this fragment to be replicated is the sequence
of two short regions of nucleotides (the subunits of DNA) at either end of the
region of interest.
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•These two short template sequences must be known so that two primers--short
stretches of nucleotides that correspond to the template sequences--can be
synthesized.
•The primers bind, or anneal, to the template at their complementary sites and
serve as the starting point for copying.
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•DNA synthesis at one primer is directed toward the other, resulting in
replication of the desired intervening sequence.
•Also needed are free nucleotides used to build the new DNA strands and a DNA
polymerase, an enzyme that does the building by sequentially adding on free
nucleotides according to the instructions of the template.
DNA Technology
•PCR is a three-step process that is carried out in repeated cycles.
•The initial step is the denaturation, or separation, of the two strands of the
DNA molecule.
•This is accomplished by heating the starting material to temperatures of around
95 C (203 F).
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•Each strand is a template on which a new strand is built.
•In the second step the temperature is reduced to about 55 C (131 F) so that the
primers can anneal to the template.
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•In the third step the temperature is raised to about 72 C (162 F), and the DNA
polymerase begins adding nucleotides onto the ends of the annealed primers.
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•At the end of the cycle, which lasts about five minutes, the temperature is
raised and the process begins again.
•The number of copies doubles after each cycle.
•Usually 25 to 30 cycles produce a sufficient amount of DNA.
DNA Technology
•In the original PCR procedure, one problem was that the DNA polymerase had to
be replenished after every cycle because it is not stable at the high
temperatures needed for denaturation.
•This problem was solved in 1987 with the discovery of a heat-stable DNA
polymerase called Taq,
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•an enzyme isolated from the thermophilic bacterium Thermus aquaticus, which
inhabits hot springs.
•Taq polymerase also led to the invention of the PCR machine.
•Because DNA from a wide range of sources can be amplified, the technique has
been applied to many fields.
DNA Technology
•PCR is used to diagnose genetic disease and to detect low levels of viral
infection.
•In forensic medicine it is used to analyze minute traces of blood and other
tissues in order to identify the donor by his genetic "fingerprint."
DNA Technology
•Within the last century, detectives increasingly have turned to scientific
evidence to help solve crimes.
•The distinctiveness of the human fingerprint was first described in 1892, the
same year the legendary Sherlock Holmes was created.
DNA Technology
•Soon to follow were the techniques for ABO blood typing and leukocyte antigen
tissue typing.
•DNA fingerprinting, first introduced in U.S. courts in 1988, was considered to
be the greatest forensic advance since classical fingerprinting.
DNA Technology
•Due to the O.J. Simpson murder trial, former topics such as the polymerase
chain reaction and restriction fragment length polymorphisms are found even in
popular news magazines.
DNA Technology
•DNA typing is based on an observation made in 1984 by Sir Alec Jeffreys.
•Jeffreys found something rather odd in the non-coding region of the human
genome: multiple copies of short nucleotide sequences, 3 to 30 base pairs long,
repeated one after another 20 to 100 times [e.g., GACTGACTGACT].
DNA Technology
•These groups of repeat sequences, called minisatellites or VNTRs (variable
number of tandem repeats), are now known to be widely scattered throughout the
human genome.
•Everyone has these repeat units in their DNA, but the number of these regions
at different loci are different in each individual.
DNA Technology
•Only identical twins end up with the same numbers and patterns of VNTR’s.
•Although DNA molecules from different individuals are more alike than they are
different, there are areas of the human genome that exhibit a great deal of
diversity.
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•Such regions are called hypervariable or "polymorphic" (meaning many forms).
•These regions provide the markers for genetic disease diagnosis and DNA typing.
•Many DNA polymorphisms are found in the parts of the genome that do not code
for protein.
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•In humans, this non-coding DNA constitutes approximately 95 percent of the
genome!
DNA Technology
•DNA polymorphisms are divided into two categories:
•sequence polymorphisms, such as occur within the genes of the human leukocyte
antigen (HLA) complex; and
•length polymorphisms, exemplified by the VNTR loci.
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•In the case of VNTRs, the polymorphism is the difference in the number of times
the sequence is repeated.
•For example, an individual heterozygous for a VNTR locus may have 3 copies of
the repeat on one chromosome and 7 copies, at that same position, on the
homologous chromosome.
DNA Technology
•Thus, when a DNA sample is cut with an endonuclease (one that does not cleave
the repeat unit) and probed with a radioactive VNTR, the length of the resultant
restriction fragment is a function of the number of copies of the tandem repeats
within the fragment.
DNA Technology
•The number of repeat units can range from a few to a few hundred, so that any
VNTR locus can exist in one of several forms, or alleles.
•An allele is one of two or more alternative forms of a single genetic locus
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•Since there are many different numbers of repeat units for any one locus among
different people, the fragment pattern revealed by analysis of multiple VNTR
loci constitutes a nearly unique genetic profile for every individual.
DNA Technology
•RFLP Technology
•Currently, the most powerful DNA profiling technique is the RFLP (restriction
fragment length polymorphism) method.
•At many crime scenes, sufficient quantities of DNA can be recovered from dried
blood spots or semen samples to make this analysis possible.
DNA Technology
•DNA is extracted from the evidence and digested with a restriction enzyme.
Fragments are separated by gel electrophoresis, denatured, and transferred onto
a nitrocellulose membrane by a process called Southern blotting.
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•The membrane is bathed in a solution containing a radioactively labeled "probe"
or single-stranded DNA molecule containing VNTR locus sequences.
•Probe molecules hybridize with complementary sequences in the immobilized
fragment, which can be visualized on X-ray film.
DNA Technology
•Forensic investigations commonly use probes for four or five different loci on
a single sample to improve the validity of the test.
•The entire process may take two months to complete; a nerve-wracking time for
defendant, prosecutors and defense attorneys.
DNA Technology
•The final DNA fingerprint is a pattern on X-ray film of light and dark bands
similar to the bar codes found on retail goods.
•Homozygotes will show one dark band rather than two lighter ones since they
have received the same DNA sequence from each parent.
DNA Technology
•Although the techniques are standard practice in many laboratories, great care
must be taken in carrying out DNA typing tests.
•Forensic samples of DNA are rarely pure. DNA from bacteria or fungi may show up
in the fingerprint; dyes from denim can interfere with restriction enzymes;
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•and proteins in the evidence sample can retard the migration of DNA fragments
in gels, a problem known as band shift.
DNA Technology
•PCR Technology
•If the forensic sample is too minuscule for RFLP testing, or if the DNA is
degraded, the polymerase chain reaction (PCR) can be used to obtain a DNA
profile.
•PCR is a molecular copying process used to amplify specific DNA sequences.
DNA Technology
•With this technique it is possible to obtain enough DNA from a single hair
follicle or a single sperm cell to determine an individual's DNA profile.
•The remarkable sensitivity of PCR is the procedure's main advantage.
•Beginning with a single molecule of DNA, thirty cycles of PCR can generate 100
billion molecules in one afternoon!
DNA Technology
•PCR can be used to selectively amplify DNA fragments containing either length
or sequence polymorphisms.
•Sequence polymorphisms, such as occur within the genes of the highly
polymorphic HLA complex, are the result of single nucleotide base changes.
DNA Technology
•Length polymorphisms are exemplified by the variable number of tandem repeat
loci (VNTR).
•Length variation at a given VNTR locus is detected by sizefractionation of PCR
products in gels.
•PCR-amplified DNA products can be directly visualized by staining after
electrophoresis, eliminating the need for radioactive probes.
DNA Technology
•the Odds
•If the DNA profile of crime scene evidence does not match the profile of the
suspect, the suspect is completely exonerated.
•DNA techniques have proven extremely useful in excluding suspects.
DNA Technology
•The FBI finds exclusions in about 30 percent of the comparisons it carries out,
making DNA-typing a major source of protection for the innocent.
•However, the converse is not true with absolute certainty.
•Thus, if DNA profiles from the evidence and a suspect are judged to match, the
strength of this evidence is measured by a "match probability,"
DNA Technology
•…the likelihood that an individual chosen randomly from an appropriate
population will match the crime profile.
•A good deal of controversy has centered on the methods for calculating this
match probability.
DNA Technology
•Our DNA comprises about 3 billion base pairs and, with the exception of
identical twins, the DNA of any two individuals differs greatly, probably by
over a million base differences.
•There is, therefore, sufficient information in the DNA to identify the culprit
unequivocally from a DNA sample recovered from a crime scene .
DNA Technology
•However, DNA typing methods analyze only a tiny fraction of all the potentially
variable sequences within the genome.
•Thus, DNA typing does not identify an individual, but rather results in a high
probability of identification.
•DNA Goes to Court
LYNN ELWELL, PhD
•North Carolina Biotechnology Center,
Research Triangle Park, North Carolina 27709
DNA Technology
•These techniques were first developed around 1975 for basic research in
bacterial molecular biology, but this technology has also led to many important
discoveries in basic eukaryotic molecular biology.
DNA Technology
•Such discoveries resulted in the appearance of the biotechnology industry.
•Biotechnology refers to the use of living organisms or their components to do
practical tasks such as:
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•The use of microorganisms to make wine and cheese
•Selective breeding of livestock and crops
•Production of antibiotics from microorganisms
•Production of monoclonal antibodies
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•The use of recombinant DNA techniques allows modern biotechnology to be a more
precise and systematic process than earlier research methods.
•· It is also a powerful tool since it allows genes to be moved across the
species barrier.
DNA Technology
•Using these techniques, scientists have advanced our understanding of
eukaryotic molecular biology.
•The Human Genome Project was an important application of this technology.
•This project's goal was to transcribe and translate the entire human genome in
order to better understand the human organism.
DNA Technology
•A variety of applications are possible for this technology, and the practical
goal is the improvement of human health and food production.
DNA Technology
•Before 1975, transferring genes between organisms was accomplished by
cumbersome and nonspecific breeding procedures.
•The only exception to this was the use of bacteria and their phages.
•Genes can be transferred from one bacterial strain to another by the natural
processes of transformation, conjugation or transduction.
DNA Technology
•· Geneticists used these processes to carry out detailed molecular studies on
the structure and functioning of prokaryotic and phage genes.
•· Bacteria and phages are ideal for laboratory experiments because
they are relatively small, have simple genomes, and are easily propagated.