Download DNA sequencing

Advanced
Biotechnology Techniques
Chapter 14
Learning Outcomes
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Discuss the benefits and implications of knowing the DNA
sequences for humans and other organisms
Explain how DNA is sequenced using the Sanger Method and the
recent improvements that have increased the efficiency of this
process
Describe some of the tools used in genomics and the advances
made possible by them
Discuss the field and potential of proteomics and the methods
used for protein study
Outline the important application of the growing biotechnology
fields for pharmacogenetics, environmental and marine
biotechnology, and bioterrorism/biodefense
14.1 Advanced DNA Topics – DNA Sequencing
DNA sequencing, polymerase chain reaction (PCR), microarray, and
bioinformatics have provided so much data that researchers must
design, conduct, and report the results of their experiments in ways that
are different from those that were standard just a generation ago.
DNA Sequencing
DNA sequencing includes all the techniques used to determine the
order of nucleotides (A, C, G, and T) in a DNA fragment.
Vocabulary
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DNA sequencing – pertaining to all the techniques that lead to determining
the order of nucleotides (A, C, G, and T) in a DNA fragment
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Dideoxynucleotide sequencing – a sequencing method that uses ddNTP and
dNTPs in a predictable way to produce synthesis fragments of varying length;
also called the Sanger Method
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Dideoxynucleotides – nucleotides that have an oxygen removed from carbon
number 3, abbreviated ddNTPs
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BLAST – an acronym for Basic Local Alignment Search Tool, a program that
allows researchers to compare biological sequences
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Cycling sequence – a technique developed in the late 1990s that allowed
researchers to run synthesis reactions over and over on samples, increasing the
amount of sequencing product and the speed of getting results
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Human Genome Project – a collaborative 10-year project completed in 2000,
which aimed to sequence the entire DNA code for the human organism
14.1 Review Questions
1.
How is ddNTP different from a “regular” dNTP?
2.
When preparing sequencing-reaction tubes, each of the four dNTPs are
added, but just one kind of ddNTP. Which are used in the highest
concentrations, the dNTPs or the ddNTPs, and why?
3.
Where on the Internet may one go to compare DNA sequence data?
4.
What additional instrument is required for cycle sequencing?
14.2 Advanced DNA Studies – Genomics
Genomics is the study of an organism’s genome, meaning all the genes
and other noncoding sequences.
RNA and Genomics
There are several forms of RNA interacting with DNA and other
RNA molecules to turn some genes on and off.
RNA of Interest
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RNAi
siRNA
microRNA
Vocabulary
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Genomics – the study of all the genes and DNA code of an organism
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Bioinformatics – the use of computers and databases to analyze and relate
large amounts of biological data
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Shotgun cloning – a method of cloning used during the sequencing of the
human genome that involves digesting DNA into 500 bp pieces, generating
libraries from those fragments, and eventually sequencing the libraries
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RNAi – abbreviation for RNA interference, a type of double-stranded RNA that
is chopped in small pieces then engulfed by the cell, and binds to and interferes
with the cells native RNA or DNA, blocking protein production
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siRNA – abbreviation for short-interfering RNA, a type of single-stranded RNA
oligo (fragment) that is created by scientists to target a gene for silencing
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microRNA – small pieces of RNA that are known to interrupt posttranscriptional
RNA function by binding to mRNA as soon as it is made
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Northern blot – a process in which RNA fragments on a gel are transferred to
a positively charged membrane (a blot) to be probed by labeled cDNA
14.2 Review Questions
1.
How much of the human genome was sequenced using shotgun
cloning?
2.
Name a plant and an animal whose entire genome has been put on a
GeneChip® microarray for the purpose of studying gene function and
expression.
3.
MicroRNA, RNAi, and siRNA all operate in a similar fashion. What do
they do?
14.3 Advanced Protein Studies
A single gene can encode for as many as 50 different protein forms.
X-Ray Crystallography
Protein (x-ray) crystallography visualizes the positions of atoms in a
molecule based on x-ray wave diffraction, and the constructive and
destructive interference that results off the atoms in the sample.
Mass Spectrometry
The mass spectrometer is an instrument that measures the masses
and relative concentrations of atoms and molecules.
Nuclear Magnetic Resonance
Nuclear magnetic resonance measures the spin on nuclei (protons)
of particular isotopes in a magnetic field.
ELISA
ELISA is one of the most frequently used assays in protein manufacturing.
Western Blots
Running samples on acrylamide gels can provide a great deal of information
about the proteins in a sample.
Protein Arrays
Protein arrays are a fusion of microarray and ELISA techniques, processed
on a “protein chip.”
Vocabulary
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Proteomics – the study of how, when, and where proteins are used in cells
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Proteome – all of an organism’s protein and protein-related material
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NMR – abbreviation for nuclear magnetic resonance, a technique that measures the spin of
nuclei (protons) of isotopes in a magnetic field to study physical, chemical, and biological
properties of proteins, including their structure in aqueous (watery) solutions
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Protein (x-ray) crystallography – a technique that uses x-ray wave diffraction patterns
to visualize the positions of atoms in a protein molecule to reveal its three-dimensional
structure
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X-ray diffraction pattern – a pattern of light intensities that develops when an x-ray
beam is passed through a mounted crystalline structure
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Salting out – a technique for crystallizing proteins that involves precipitating a sample of
pure protein using a stringent salt gradient of sodium chloride, ammonium sulfate, or some
other salt
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Protein arrays – a fusion of technologies, where protein samples bound on the glass slide
(protein chip) are assessed using antibodies or other recognition material
14.3 Review Questions
1.
What is the technique called that uses an x-ray to study the structure
of protein crystal? What is the picture called?
2.
What causes proteins to crystallize out of solution?
3.
What binds to what during an ELISA? How can a technician know that
a protein is present during an ELISA?
4.
What binds to what during a Western blot? How can a technician know
that a protein is present during a blot?
14.4 Other Advances and Applications of Biotechnology
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Pharmacogenetics
Environmental biotechnology
Biodefense
Pharmacogenetics – Personalized Medicines
Since every person’s DNA is different, doctors will be able to design and
modify drugs to better meet individual needs.
Environmental Biotech
This is a vast field with many applications for monitoring and correcting the
health of entire species, populations, communities, and ecosystems.
Biodefense: Protection Against Bioterrorism
Biodefense is the term for all of the methods used to protect people from
exposure to biological agents.
Vocabulary
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Pharmocogenetics – a branch of biotechnology that involves utilizing the genetic and
protein codes to design or improve medications
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Environmental biotechnology – a field of biotechnology whose applications include
monitoring and correcting the health of populations, communities, and ecosystems
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Marine biotechnology – the study and manipulation of marine organisms, their
component molecules, cells, tissues, or organs
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Bioterrorism – the use of biological agents to attack humans, plants, or animals
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Biodefense – relating to all the methods used to protect a population from exposure to
biological agents
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Nanotechnology – pertaining to all technologies that operate on a nanometer scale
14.4 Review Questions
1.
What is the name of the process by which strategies are used to solve
environmental problems, such as oil spills, soil erosion, or fertizier
pollution?
2.
What is the function of the Autonomous Pathogen Detection System?
3.
What is the approximate size of the instruments and products of
nanotechnology?
4.
Give an example of a personalized medicine.
Questions and Comments?