Download dna technology chapter 20

DNA TECHNOLOGY
CHAPTER 20
Campbell
Chapter 13 Mader
What is it?
• DNA technology is the chemical
manipulation of the genotypes and
resulting phenotypes of organisms such
that living organisms are modified;
alternatively, no-longer-living organisms or
their no-longer-living parts may be
analyzed chemically at the level of
genotype
Why important?
• The use of DNA technology has
revolutionized how scientists study the
genetics, biochemistry, even the ecology
and evolutionary biology of organisms,
plus has allowed the development of novel
biological products, indeed whole
industries are now devoted to DNAtechnology-based production and analysis
of biological materials
Genetic Engineering is…
• the artificial manipulation of the genetic
material of organisms, including the
creation of novel genetic material (i.e.,
novel nucleotide sequences)
• This manipulation occurs to a large extent
external to organisms, e.g., in test tubes,
a.k.a., in vitro (meaning, literally, “in
glass”)
Genetic Engineering is used to…
• Make recombinant DNA
• To purposefully change
nucleotide sequences
• To clone DNA
-manipulation of an organism’s genotype by
artificial, typically very direct means
Biotechnology is …
– Contrasting with genetic engineering,
biotechnology refers to the engineering of
phenotype
– Biotechnology is not limited to the
manipulation of phenotype by directly
manipulating genotype (i.e., via genetic
engineering) though today this is very often
how phenotypes are manipulated (often to
the detriment of more traditional means such
as plant and animal breeding)
Molecular techniques – there are
many!
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Gene cloning - rDNA (and is associated with
various molecular techniques—including in vitro
restriction enzyme digests, and DNA ligation—
plus additional, less-artificial manipulations
including transformation and transduction)
Creation of cDNA
Polymerase chain reaction
Gel electrophoresis
Various blotting techniques (Southern blotting,
Northern blotting, Western blotting, etc.)
RFLP analysis
DNA sequencing
Isolating DNA
• fairly straightforward involving the breaking
open of cells and subsequent purification
of the DNA component
• The actual molecular manipulation of DNA
begins only once the DNA is purified, and
involves to a large extent the cutting of
DNA at specific nucleotide sequences by
proteins known as restriction enzymes
Restriction Enzymes and the
Restriction Fragment
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The actual nucleotide sequence on a piece of
DNA that a restriction enzyme cuts is called a
restriction site
Most restriction sites are palindromes with
identical sequences regardless of the direction
one moves down the DNA (keeping in mind, of
course, that DNA is antiparallel such that one
moves down or up a different strand if one
switches direction : What are some
palindromes in English language?
How the RE cuts
• RE are used (and harvested) for their
specific cutting ability
• In order to make rDNA – need nice sticky
end but blunt ends are produced too
Restriction fragment length
polymorphism (RFLP)
• The location of restriction sites in a
genome occurs fairly randomly, and can
differ from person to person
• differences in nucleotide sequences at
specific loci found within populations
polymorphisms
RFLPs Importance
• As a consequence of these differences between
individuals in the location of specific restriction
sites, the distance between sites will vary, thus
length of restriction fragments produced by
digesting an individual’s genome using specific
restriction enzymes will also vary
• The variation between individuals is called
restriction fragment length polymorphism
Remember OJ – the glove!
• Since the nucleotide sequence of nearly
every individual is unique, the RFLPs of
each individual are also unique, and thus
RFLP analysis may be employed to
forensically distinguish individuals (hence
the synonymous term, DNA fingerprinting)
Comparing the band patterns!
Gel Electrophoresis
• Gel electrophoresis is a method of
molecule (or complex) separation in which
molecules move within a gel-like medium
(think jello or, even better, the agar found
in a petri dish)
Gel Electrophoresis Continued
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In gel electrophoresis charged molecules
are pulled through the gel (typically
consisting of the polymer polyacrylamide or
of purified agar known as agarose)
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Larger or more diffuse molecules move more
slowly because they tend to get hung up in the
gel matrix
Molecules with greater charges also move faster
because it is an electric voltage that is employed
to pull the molecules through the gel matrix
OVERVIEW
• http://web.utk.edu/~khughes/GEL/sld001.h
tm
Gene Cloning –the steps
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Isolating DNA from the cell of an organism
(including digestion with restriction enzymes)
Insertion of that DNA into a plasmid
Placement of the plasmid into a second cell
Measures taken to make sure that the cloned
DNA is the DNA of interest
Various manipulations of the DNA including
subcloning, sequencing, and expression
How to make rDNA – remember we
need those sticky ends
• http://www.biology87.org/apbio/molgen/Act
ivity6_notes.pdf
Many purposes for rDNA
• Figure 20.1
– Protein harvesting
– Alter genetic make-up
– Resistance
– Cloning a gene of interest (fig. 20-3)
PCR= Polymerase Chain Reaction
• In polymerase chain reaction, one
employs short DNA primers that are
complementary to the opposite ends of a
specific sequence of DNA one in
interested in amplifying in number (again,
keep in mind that DNA is antiparallel and
that consequently opposite ends means
also complementary to opposite strands)
PCR continued
• The primers supply the 3’ –OH “primer”
necessary for the initiation of DNA
replication
• Polymerization is used to produce doublestranded DNA (i.e., a double helix) using
single-stranded DNA as the template
Still PCR
– Individual single-strands of DNA are typically
formed via the unwinding of a DNA double
helix by the application of heat
– Thus, PCR consists of heat treatment to
unwind DNA, binding of primers to the
resulting single-stranded DNA,
polymerization of new DNA to form a new
double-stranded DNA double helix, repeat
See Methods: The Polymerase Chain Reaction
(PCR) found on page 371
• Neat things about PCR
• Can be performed in a single reaction
vessel to which one has to add the various
necessary ingredients only once—thereby
repeated rounds of synthesis may be
effected simply by heating and cooling the
reaction vessel
Another neat thing about PCR
• may be initiated using only very small,
impure samples of DNA
-as in dinosaur DNA, or teeny tiny
samples from a crime scene (even partial
pieces)
Southern Blotting
• Combo of methods
– DNA isolation and _________________________
– DNA run using ____________________________
– _____________ - use alkaline solution and sponge to
pull DNA fragments onto nitrocellulose paper,
denatures the DNA into single strands
– _________________________________ –
radioactive single stranded DNA that is
complementary to the gene of interest – a solution of
these probes
– ____________________________ - photo image of
the radioactive bands ONLY!
WHY USED?
• http://www.dnalc.org/ddnalc/resources/sho
ckwave/southan.html
Watch animation and summarize how it is used in forensics:
SANGER METHOD
• Used to determine___________________
• How it works:
– One piece of DNA is divided into ___ portions
– Each portion is placed in a mixture that
contains all enzymes and primers needed to
synthesize cDNA
– Key difference between TT,
__________________________________
Sanger Continued
– Each TT contains the same DNA but
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TT1 – radioactive ___’s
TT2 – radioactive ___’s
TT3 – radioactive ___’s
TT4 – radioactive ___’s
- The marked DNA are run using highly
specific agarose that separates the DNA
into __________________________.
Reading the nucleotide sequence
• http://www.zerobio.com/sanger1.htm
You try it!
• What is the DNA sequence?
Practical Applications – present
Tuesday
• Medical Contributions
– Diagnosis of disease
– Human Gene Therapy
– Pharmaceuticals
• Forensics, Environmental, Agriculture
– CSI
– Environmental
– Agriculture (3 people)
• Safety and ethical issues
TEST on Chapter 20, parts of 18
and 19 on Weds.
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Mon – finish biotech techniques
Tues – quick presentations on Apps.
Weds – Test on 18, 19 and 20!
Thursday – Bioterrorism Holiday Party!
» Half-year review guide
» Essay practices – three will be selected for the final
exam. Write outline for all of them.