4. DNA SEQUENCING
The ultimate level of analysis is determination
of the actual sequence of bases in a DNA
molecule. The development of sequencing
has paralleled the advancement of molecular
biology. The genomics was born out of the
ability to determine the sequence of an entire
genome relatively rapid.
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5. Idea
To generate a set of nested fragments that
each begin with the same sequence and end
in a specific base. When this set of fragments
is separated by high resolution gel
electrophoresis, the results is a “ladder” of
fragments in which each band consist of
fragments that end in a specific base. By
starting with the shortest fragments, one can
read the sequence by moving up the ladder.
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6. ENZYMATIC SEQUENCING
Developed by Fredrick Sanger, who also was
the first to determine the complete sequence
of a protein.
This method uses dideoxynucleotides as chain
terminators in DNA synthesis reactions. A
dideoxynucleotides has H in place of OH at
both the 2’ position and at the 3’ position.
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7. AUTOMATED SEQUENCING
The technique of enzymatic sequencing is
very powerful, but it is also labor intensive and
takes a significant amount of time. It requires
a series of enzymatic manipulations, time for
electrophoresis, then time to expose the gel to
film.
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9. Review: The structure of DNA
Helix
Complementary Base Pairing 9
Chapter 5 : DNA TECHNOLOGY
10. Review: The structure of DNA
Unzipping
Antiparallel Strands
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11. Review: Genome Sizes
Pine: 68 billion bp
Corn: 5.0 billion bp
Soybean: 1.1 billion bp
Human: 3.4 billion bp
Housefly: 900 million bp
Rice: 400 million bp
E. coli: 4.6 million bp
HIV: 9.7 thousand bp
11
Chapter 5 : DNA TECHNOLOGY http://www.cbs.dtu.dk/databases/DOGS/abbr_table.txt
12. Just How Big Is 3.4 Billion?
Human genome is 3.4 B bp
If the bases were written in standard
10-point type, on a tape measure...
...The tape would stretch for 5,366
MILES!
Identifying a 500bp sequence in a
genome would be like finding a
section of this tape measure only 4
feet long!
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13. The Problem...
How do we identify and detect a specific
sequence in a genome?
TWO BIG ISSUES:
There are a LOT of other sequences in a genome
Y
ICIT that we’re not interested in detecting.
ECIF
SP
The amount of DNA in samples we’re interested in is
ION
TVERY small.
A
FIC
PLI
AM
PCR solves BOTH of these issues!!!
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14. PCR
- a method for amplifying (copying) small
amount of DNA in nearly any amount
required, starting with a small initial quantity.
- an in vitro or cell-free method for
synthesizing DNA.
- it was invented in 1985 by Kary Mullis
(received the Nobel Prize for chemistry in
1993).
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15. PCR History
In what has been called by some the greatest achievement of
modern molecular biology, Kary B. Mullis developed the
polymerase chain reaction (PCR) in 1983. PCR allows the
rapid synthesis of designated fragments of DNA. Using the
technique, over one billion copies can be synthesized in a
matter of hours.
PCR is valuable to scientists by assisting gene mapping, the
study of gene functions, cell identification, and to forensic
scientists in criminal identification. Cetus Corporation, Mullis'
employer at the time of his discovery, was the first to
commercialize the PCR process. In 1991, Cetus sold the PCR
patent to Hoffman-La Roche for a price of $300 million. It is
currently an indispensable tool for molecular biologists and
the development of genetic engineering.
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16. Idea
Simple
Two primers are used that complimentary to the
opposite strands of a DNA sequence, oriented
toward each other. When DNA polymerase acts
on these primers and the sequence of interest,
the primers produce complementary strands,
each containing the other primer. If this
procedure is done cyclically, the result is a large
quantity of a sequence corresponding to the
DNA that lies between the two primers.
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19. PCR
Components of PCR
Template DNA
primers
dNTPs (dATP, dTTP, dCTP & dGTP)
Taq DNA polymerase
MgCl
2
PCR buffer, pH 8
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20. PCR
Three major phases in PCR:
Denaturing –high temperature (94ºC)
Annealing of primers –low temperature(55ºC)
Extension – synthesis – intermediate
temperature (72ºC)
The total time to perform a standard PCR is
approximately 4 hours.
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23. Factors influencing PCR
Quality of template DNA
Concentration of template DNA
Primers
Concentration of MgCl2
Annealing temperature
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24. Quality of template DNA
- should be free of proteases that could
degrade the DNA polymerase.
- template DNA with high levels of
proteins or salts should be diluted or
cleaned up to reduce inhibition of DNA
polymerase activity.
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25. Concentration of template DNA
- highly concentrated template DNA may
yield nonspecific product or inhibit the
reaction.
- it is rare that template DNA concentration
is too low.
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26. Primers
- select primers with a random base
distribution and GC content similar to
template DNA being amplified.
- avoid sequences with secondary
structure, especially at the 3’ end.
- check primers for complementary and
avoid primers with 3’ overlaps to reduce
primer-dimer artifacts.
- design so the base at the 3’ end of the
primer is a G or C to enhance specificity.
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27. Concentration of MgCl2
- MgCl2 concentration is very important.
- excess Mg2+ promotes production of
nonspecific product and primer-dimer
artifacts.
- insufficient Mg2+ reduces yield.
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28. Annealing temperature
- annealing temperature depends on length
and GC content of primers (55ºC good for
primers 20 nucleotides long; 50%).
- Higher annealing temperatures may be
needed to increase primer specificity.
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29. Some Uses of PCR
Forensic DNA detection
Identifying transgenic plants
Detection and quantification of
viral infection
Cloning
Detection of ancient DNA
Gene expression analysis
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30. Uses of PCR: Ancient DNA
Archaeologists have happily seized on PCR
and are applying it in an amazing variety of
ways. It is helping, for example, to launch a
new chapter in the colorful and controversial
story of the 2000-year-old Dead Sea Scrolls,
which are written on parchment made out of
skins from goats and gazelles. Researchers are
analyzing the parchment fragments to try to
identify individual animals they came from.
The hope is that the genetic information will
guide them in piecing together the 10,000
particles of scrolls that remain.
Chapter 5 : DNA TECHNOLOGY
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31. Uses of PCR: Disease Detection
PCR can also be more accurate than standard tests.
It is making a difference, for example, in a painful,
serious, and often stubborn misfortune of
childhood, the middle ear infection known as otitis
media. The technique has detected bacterial DNA
in children's middle ear fluid, signaling an active
infection even when culture methods failed to
detect it. Lyme disease, the painful joint
inflammation caused by bacteria transmitted
through tick bites, is usually diagnosed on the basis
of symptom patterns. But PCR can zero in on the
disease organism's DNA contained in joint fluid,
permitting speedy treatment that can prevent
serious complications.
Chapter 5 : DNA TECHNOLOGY
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32. Uses of PCR: Endangered Species
Researchers have used the technique to aid in
reducing illegal trade in endangered species, and
products made from them. Because PCR is a
relatively low-cost and portable technology, and
likely to become more so, it is adaptable for field
studies of all kinds in the developing countries. It is
also a tool for monitoring the release of genetically
engineered organisms into the environment.
Chapter 5 : DNA TECHNOLOGY
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33. Uses of PCR: Forensic DNA
The technique's unparallelled ability to identify and
copy the tiniest amounts of even old and damaged
DNA has proved exceptionally valuable in the law,
especially the criminal law. PCR is an
indispensable adjunct to forensic DNA typingcommonly called DNA fingerprinting.
Chapter 5 : DNA TECHNOLOGY
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34. Uses of PCR: Proving Innocence
DNA typing is only one of many pieces of evidence
that can lead to a conviction, but it has proved
invaluable in demonstrating innocence. Dozens of
such cases have involved people who have spent
years in jail for crimes they did not commit. Many
people have been freed because of the power of
PCR. Even when evidence such as semen and
blood stains is years old, PCR can make unlimited
copies of the tiny amounts of DNA remaining in
the stains for typing.
Chapter 5 : DNA TECHNOLOGY
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35. Uses of PCR: Disease Detection
The method is especially useful for searching out
disease organisms that are difficult or impossible to
culture, such as many kinds of bacteria, fungi, and
viruses, because it can generate analyzable
quantities of the organism's genetic material for
identification. It can, for example, detect the AIDS
virus sooner during the first few weeks after
infection than the standard ELISA test. PCR looks
directly for the virus's unique DNA, instead of the
method employed by the standard test, which looks
for indirect evidence that the virus is present by
searching for antibodies the body has made against
it.
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36. Uses of PCR: Ancient DNA
Archaeologists are finding that PCR can
illuminate human cultural practices as well as
human biology. Analyzing pigments from
4000-year-old rock paintings in Texas, they
found one of the components to be DNA,
probably from bison. The animals did not live
near the Pecos River at that time, so the paleoartists must have gone to some effort to obtain
such an unusual ingredient for their paint.
Taking so much trouble suggests that the
paintings were not simply decorations, but had
religious or magical significance.
Chapter 5 : DNA TECHNOLOGY
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37. Uses of PCR: Disease Detection
PCR can even diagnose the diseases of the past.
Former vice president and presidential candidate
Hubert H. Humphrey underwent tests for bladder
cancer in 1967. Although the tests were negative,
he died of the disease in 1978. In 1994, researchers
compared a 1976 tissue sample from his cancerridden bladder with his 1967 urine sample. With
the help of PCR amplification of the small amount
of DNA in the 27-year-old urine, they found
identical mutations in the p53 gene, well-known for
suppressing tumors, in both samples. "Humphrey's
examination in 1967 may have revealed the
cancerous growth if the techniques of molecular
biology were as well understood then as they have
become," the researchers said.
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38. Uses of PCR: Gene Expression Analysis
The Human Genome Project has identified tens of
thousands of genes in the human genome. A key
questions is: what do these genes do? Part of the
answer comes from determining when the genes are
turned on and off, and what affects the level of
gene expression. Quantitative PCR is a key
component of determining the levels of gene
expression, and is a critical tool in cancer research,
disease studies, and developmental biology.
DNA
RNA
GENEX Analysis
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Enzymes
Biology
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