Genome technology

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Genomics
Genomics is the study of all nucleotide sequences for an organism of interest,
including genes, pseudogenes, noncoding regions, and regulatory regions.
In human genomics, identifyingthesequenceof theentireset of chromosomes was
a major achievement. The human genome sequence was determined by first
forming DNA libraries, sequencing each of the clones, and finally ordering the
sequences using computer algorithms, genetic maps, and physical maps.
Genetic maps are based on the relative order of genetic markers, but the actual
distancebetween themarkers is hard to determine. Physical maps aremore precise
and give the distance between markers in base pairs.
Determining the order of various markers makes genetic maps. Some markers
include RFLPs, SNPs, VNTRs, and microsatellite polymorphisms.
Without thegreat advances in computing, theHuman GenomeProject would have
taken much longer and cost more money. Data mining on the information has
identified many potential protein coding regions, regulatory elements, and
different types of repetitive elements. The human genome is predicted to contain
about 25,000 genes, SINES, LINEs, and tandem repeats such as telomeres,
centromeres, and satellite DNA. Computer analysis of these complex sequences is
called bioinformatics.
Genomics has changed many different fields of study, including medicine,
evolutionary biology, and pharmacology. Understanding and identifying new
genes related to diseases has changed the way new diseases are treated and
diagnosed. Much of the textbook is devoted to these advances.
Gene testing is the most common present application. Once genes have been
associated with particular diseases, people can be screened for genetic mutations
within the gene. Such tests can diagnose diseases such as muscular dystrophy,
cystic fibrosis, sickle cell anemia, and Huntington’s disease because these are
strictly inherited disorders. In diseases with an environmental component, genetic
testing offers information that may change how a person lives his or her life.

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Perhaps those with a genetic predisposition to colon cancer will have more
screenings, earlier thanusual, and perhaps alter their diet to minimize the chance
of cancer developing. Other applications include gene therapy.
Mutations occur in all organisms at random places in the genome with an
approximately similar rate. The length of insertions and deletions is variable.
Thestudy of genomics focuses on mutations in the genome, by identifying single
nucleotidepolymorphisms, methylationpatterns, or differences in tandem repeats.
Mutations includesinglenucleotidechanges,inversions, deletions, and insertions.
PHARMACOLOGY hopeto correlatethesedifferences with drug sensitivity, thus
preventing adversedrug reactions. design and testingof pharmaceuticals.
Penicillin is one of the 20th century’s greatest discoveries, but was found by
Accident. Another problem of drug development is adverse drug reaction (ADR).
Adverse reactions may happen in some patients whileothers respond well and are
cured by the same drug. Most drugs are developed with the average patient in
mind, yet there is often a subset of people who react badly to the drug. For
example, many people are allergic to penicillin or other types of antibiotics.
Adverse drug reactions are a major cause of hospitalizations and death. The
differences in reaction to certain drugs often depend on the person’s genetic
makeup.
Pharmacogenomics is the study of all the genes that determine drug response in
humans. Morespecifically, pharmacogenetics is the study of inherited differences
in drug metabolismand response. The goal of these fields of study is to reduce the
number of ADRs by determining thegenetic makeup of the patient before offering
a specific drug. Thekey to making a “genetic” diagnosis is the use of SNPs . Single
changes in coding regions can often be correlated with adversedrug reactions. For
example, if a certain subpopulation of people does not respond to a drug, then
their DNA can be examined for a specific SNP that is absent in patients who do
respond. Before the drug is given to any new patients, DNA from a blood sample

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can be tested for the presence of that SNP. Testing for SNPs can be done by
microarray analysis in the doctor’s office, thus reducing the number of office or
hospital visits. SNP analysis is also used to screen for hereditary defects.
Specifically, SNPs can be identified using a techniquecalled Zipcode analysis. Here
many different SNPs can be examined simultaneously.
First, PCR is used to amplify the region containing each different SNP being
investigated. The PCR fragments could be sequenced in full, but because SNPs
differ by only one base, single base extension analysis is done instead. For this, a
primer is designed to anneal just one base pair away from the SNP location. This
primer also carries a “zipcode” region that is used to identify this specific SNP, and
each SNP has a different zipcode. After the Zipcode primer anneals to the PCR
fragments, DNA polymerase plus fluorescently labeled dideoxynucleotides are
added. This results in a single base being added to the primer. (Note that
dideoxynucleotides block chain elongation, and so only one base can be added.)
Each base is labeled with a different fluorescent dye, allowing it to be identified.
Next, beads linked to complementary zipcode (cZipcode) sequences are added to
grab the zipcoded primers. The trapped Zipcode primer with the labeled
nucleotide has a different color based on which base was incorporated. The
different colors can be sorted and counted by FACS or fluorescent activated cell
sorting .
One of the spin-offs from theHuman Genome Project is thePharmacogenetics and
Pharmacogenomics Knowledge Base (PharmGKB; http://www.pharmgkb.org/ ).
This records genes and mutations that affect drug response. Consider asthma, a
condition wherepeople overreact toinhaled irritants by cutting airflowin and out
of the lungs. The muscle cells around the bronchial tubes constrict, decreasing
airflow. Albuterol is a drug used to open the airways in people with asthma. This
drug opens the bronchialtubes by relaxing the muscle cells. Albuterol affects the
beta2-adrenergic receptor, and mutations in this receptor alter the efficacy of
albuterol. A single nucleotide change that replaces glycine at position 16 with
arginine gives a receptor protein with a better response to albuterol. Whether a
patient has this SNP will determine whether or not albuterol will be effective.

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Another key area of pharmacogenetics concerns the cytochrome P450 family of
enzymes. These play a role in the oxidativedegradation of many foreign molecules,
including many pharmaceuticals. The CYP2D6 isoenzyme oxidizes drugs of the
tricyclic antidepressant class, and different alleles of this enzyme affect how well a
person metabolizes these drugs. Much as for albuterol, identifying which allele a
patient has will prevent overdosages or adverse reactions. As time goes on, more
medical treatments will be designed for the individual rather than the average
person.
Pharmacogenetics is the study of inherited differences in drug metabolism and
response. Some SNPs affect how a person metabolizes a certain drug. By
determining what SNP correlates with what drug sensitivity, new patients can be
screened and possibly avoid adverse drug reactions (ADRs)

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In evolutionary biology, physical features have always determined the relatedness
of two organisms. Sincethegenomes of many organisms havebeen sequenced, the
genetic code for highly conserved genes is used to determine relatedness. Over
time, mutations accumulate within every genome. The more essential genes
change slowly over time, whereas less essential genes incorporate more changes.
Molecular phylogenetics uses genomic sequences of different organisms in order
to determine their evolutionary relatedness. Essential proteins have fewer
mutations over time. Less essential proteins have more mutations over time.
Genomics also encompasses gene expression, whichis done on a global scale using
genomic microarrays. These arrays have DNA from the genome, either a pure
cDNA or a synthesized oligonucleotide, linked to a glass slide. The fluorescently
labeled mRNA sample of interest is then hybridized to the microarray. When an
mRNA hybridizes to the immobilized cDNA or oligonucleotide, that region will
fluoresce. The amount of fluorescence correlates to the amount of mRNA in the
sample. These microarrays are very flexible and can be designed to cover the
whole genome as in whole-genome arrays, or they can be designed to a subset of
genes. In singlegene analysis, specific regulatory regions defined by genomics are
linked to a variety of different reporter genes, including β -galactosidase, alkaline
phosphatase, luciferase, and green fluorescent protein. These studies replace the
actual gene of interest with the reporter gene, but leave the regulatory regions
upstreamor downstream. Theamount of reporter gene product is a direct measure
of the strength of expression for the regulatory region under study.
DNA Vaccines
A recent development in vaccinology is immunization with polynucleotides. This
has been referred to as genetic immunizationor DNA immunization.The rationale
for this is that cells can take-up DNA and expresses the genes within the
transfectedcells. Thus, the animalbody itself produces thevaccine. This makes the
vaccine relatively inexpensive to produce. Some of the advantages of
polynucleotideimmunization arethat it is extremely safe, induces a broad rangeof
immune responses (cell-mediated and humoral responses), long-lived immunity,
and, most importantly, can induceimmune responses in the presence of maternal

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antibodies. Most recently, it has also been used for immunizing animal fetuses.
Thus, animals are born immune to the pathogens and at no time in the animal’ıs
life are they susceptible to these infectious agents. Although attractive
development, there is a great need to develop better delivery systems to improve
the in vivo e efficiency.
Reverse vaccinology
The technology has twomajor facets, in silico and in vitro/vivo. The in silico aspect
is the identification,annotation and then localization of ORFs and their products.
Identified targets can then be used for laboratory study (in vitro/in vivo) where
they are expressed, purified and tested for immunogenicity. Genome-based
vaccinediscovery was applied for the first time to serogroup B meningococcus, a
bacterium which is a major cause of sepsis and meningitis, that had defied all
traditional approaches to vaccine development.
The in silico sequence of the genome predicted 600 potential antigens. Of them
350 were expressed in Escherichia coli, purified and used to immunize mice.
Twenty nine were found to induce bactericidal antibodies, which will lead to
protection. A subgroup of the genome-derived antigens is now being tested in
clinicaltrials. Reverse vaccinology is now a standard technology. Vaccines projects
are not now undertakenwithout knowledge of the the sequence of the pathogen.
Successfulexamples of genome-based vaccinediscovery arepneumococcus,group
B streptococcus, Staphylococcus aureus, and a variety of viruses.