Important words and concepts from Chapter 20,
Campbell & Reece, 2002 (1/29/2005):
by Stephen T. Abedon (abedon.1@osu.edu)
for Biology 113 at the Ohio State University
|
|
|
Course-external links are
in brackets Click [index] to access site index Click here to
access text’s website Vocabulary
words
are found below |
|
|
(1) 
Chapter title: DNA Technology
(a)
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
(b)
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 DNA-technology-based production and analysis of
biological materials
(c)
[DNA technology (Google Search)] [index]
(a)
Genetic engineering is the artificial manipulation of the genetic
material of organisms, including the creation of novel genetic material (i.e.,
novel nucleotide sequences)
(b)
This manipulation occurs to a large extent external to organisms, e.g.,
in test tubes, a.k.a., in vitro
(meaning, literally, “in glass”)
(c)
Genetic engineering is employed to
(i)
Make recombinant DNA
(ii)
To purposefully change nucleotide sequences
(iii)
To clone DNA
(d)
In short, genetic engineering represents the manipulation of an
organism’s genotype by artificial, typically very direct means
(e)
[genetic engineering
(Google Search) [index]
(a)
Contrasting with genetic engineering, biotechnology refers to
the engineering of phenotype
(b)
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)
(c)
[biotechnology (Google Search)] [index]
(a)
Together the manipulation and analysis associated with DNA technology, genetic engineering,
and biotechnology are based on a number of technologies generally
referred to as molecular techniques
(b)
Molecular techniques include (though are not limited to)
(i)
Gene cloning (which is associated with various molecular
techniques—including in vitro restriction enzyme
digests, and DNA ligation—plus additional, less-artificial
manipulations including transformation and transduction)
(ii)
Creation of cDNA
(iii)
Polymerase chain reaction
(iv)
Gel electrophoresis
(v)
Various blotting techniques (Southern blotting,
Northern blotting, Western blotting, etc.)
(vi)
RFLP analysis
(vii)
DNA sequencing
(c)
[molecular techniques
(Google Search)] [index]
(a)
What is molecular biology? Good question
(b)
Basically molecular biology consists of all of the above: DNA technology, genetic engineering,
biotechnology,
and molecular techniques
(c)
Some consider molecular biology to be a science; others consider
molecular biology to be a collection of techniques (e.g., as listed above)
(d)
Sometimes people use the phrase molecular biology when they really
ought to be using the phrase molecular genetics
(e)
Basically, this chapter represents an introduction to molecular biology
(while chapters 16, 17, 18, and 19 dealt with
various aspects of molecular genetics)
(f)
At least for the past 20 years or so (perhaps and then some) molecular
biology has been a hot ticket toward earning money as a biological researcher,
and more and more disciplines are jumping on the molecular biology bandwagon,
thereby making an understanding of molecular biology and molecular
techniques almost (but not quite universally) a prerequisite to employability
as a modern biological researcher; translation: if you want to do biology
and not do molecular biology, then you’ve got to be very good at (and very
dedicated to) whatever it is that you otherwise do (update, 3/12/02: even I’ve
started using molecular techniques in my research)
(g)
[molecular biology (Google Search)] [index]
(a)
Gene cloning consists of a number of molecular techniques
that ultimately serve to place a defined segment of DNA within an organism,
typically a different organism from which the DNA was originally derived, such
that the DNA segment may be replicated repeatedly within the recipient organism
(b)
To understand gene cloning, it is becoming traditional to walk students
through the steps involved in a typical application of gene cloning
(c)
See Figure 20.1, An overview
of the how bacterial plasmids are used to clone genes for biotechnology
(d)
Steps involved in gene cloning include:
(i)
Isolating DNA from the cell of an organism (including digestion with restriction enzymes)
(ii)
Insertion of that DNA into a plasmid
(iii)
Placement of the plasmid into a second cell
(iv)
Measures taken to make sure that the cloned DNA is
the DNA of interest
(v)
Various manipulations of the DNA including subcloning, sequencing,
and expression
(e)
“For cloning genes or other pieces of DNA, plasmids are first isolated
from bacterial cells. FIGURE 20.1 follows one plasmid as a foreign gene—from a
eukaryotic cell, in this example—is inserted into it. The plasmid is now a
recombinant DNA molecule combining DNA from two sources. The plasmid is
returned to a bacterial cell, which then reproduces to form a cell clone. The
foreign gene carried by the plasmid is “cloned” at the same time, for the
dividing bacterium continues to replicate the recombinant plasmid. Under
suitable conditions the bacterial clone will make the protein encoded by the
foreign gene.”
(f)
[gene cloning, cloning DNA (Google)] [index]
(a)
The actual isolation of DNA is fairly straightforward involving the
breaking open of cells and subsequent purification of the DNA component [isolation of DNA links (MicroDude)]
(b)
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
(c)
[restriction enzyme,
restriction endonuclease
(Google Search)] [index]
(8) Restriction site (restriction fragment)
(a)
The actual nucleotide sequence on a piece of DNA that a restriction
enzyme cuts is called a restriction site
(b)
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; example of a palindromes found in the English language,
sort of: “Nodeba Bob Abedon”—my alter ego; see also: Leo’s Palindrome Collection and World’s First Palindromic URL?)
(c)
“Restriction enzymes cut covalent phosphodiester bonds of both strands,
often in a staggered way, as indicated in the diagram. Since the target
sequence usually occurs (by chance) many times in a long DNA molecule, an
enzyme will make many cuts. Copies of a DNA molecule always yield the same set
of restriction fragments when exposed to that enzyme. In other words, a restriction
enzyme cuts a DNA molecule in a reproducible way.”
(d)
See Figure 20.2, Using a
restriction enzyme and DNA ligase to make recombinant DNA
(e)
[restriction site, restriction fragment
(Google Search)] [index]
(a)
Note that most restriction enzymes do not make blunt cuts
(b)
That is, upon restriction digestion, DNA will contain short
single-stranded regions at their ends
(c)
See Figure 20.2, Using a
restriction enzyme and DNA ligase to make recombinant DNA
(d)
These short regions are termed sticky ends because two DNA’s cut by the
same restriction enzyme (or even different enzymes if they produce overhangs of
the same sequence) can hydrogen bond together via nitrogenous base pairing
(e)
Thus, a double-stranded DNA (double helix) even though it has been
digested by restriction enzymes is still to some extent capable of holding
together as a cut-but-still-intact double helix
(f)
However, note that these hydrogen-bonded fragments are not strongly
bonded together, i.e., following restriction digestion the tendency is for
restriction fragments to separate and then to only transiently reattach (and if
more than one complementary sticky end is present, reattachment is not
necessarily in the same order fragments were in within the original DNA
molecule)
(g)
[sticky ends (Google Search)] [index]
(a)
Sticky ends (as well as blunt ends) may be covalently bonded
together using the enzyme DNA ligase; see figure to right à
(b)
See Figure 20.2, Using a
restriction enzyme and DNA ligase to make recombinant DNA
(c)
Recall that DNA ligase is normally employed by cells during DNA replication
(as well as during the repair of DNA damage)
(d)
[DNA ligase (Google Search)] [index]
(a)
Once the DNA coming from two different organisms has been ligated
together, we can now call it recombinant DNA
(b)
[recombinant DNA (Google Search)] [index]
(a)
Ligating restriction fragments
together in a useful way and order is as much a science as it is an art (heck,
it’s engineering: genetic engineering)
(b)
When cloning DNA a typical step that follows DNA isolation and restriction digest is the insertion of that DNA
(i.e., ligation) into a cloning vector
(c)
Cloning vectors are typically plasmids and may
also be the partial genomes of phages
(d)
The idea behind a cloning vector is that it is a piece of DNA whose job
it is to carry pieces of DNA isolated from one organism into another organism
where that DNA (along with the vector in which it is contained) is then
replicated and the piece of DNA from the first organism may be expressed
(e)
See Figure 20.3, Cloning a
human gene in a bacterial plasmid: a closer look
(f)
Note that the vector is also subject to restriction digestion before
the insertion of foreign DNA; this creates an insertion point that, ideally,
possesses the same or similar sticky ends to those possessed by the
restriction-digested foreign DNA—thus the foreign DNA may hydrogen bond into
the cloning vector and then may be bonded covalently following incubation in
the presence of DNA ligase
(g)
Note that cloning vectors can be quite sophisticated, possessing
various means by which bacteria that contain the vector may be positively
selected from bacteria that do not (e.g., via the use of antibiotic resistance
genes) as well as various means by which the presence of an inserted fragment
may be visualized (e.g., by the disruption of a copy of the lacZ gene, which codes for b-galactosidase, the enzyme that allows
Escherichia coli to digest the sugar lactose)
(h)
Cloning vectors are taken up by cells by transformation or by transduction
(i)
[cloning vector (Google Search)] [index]
(13) Identification of the proper clone
(a)
One of the most difficult steps in gene cloning is
making sure that one has succeeded in cloning the desired DNA
(b)
First, one has to make sure that the organism being cloned has
successfully taken up the cloning vector (e.g., via the presence of
antibiotic resistance genes in cloning vectors and antibiotics in growth media—all
bacteria not-possessing the cloning vector will be killed by the antibiotic,
thereby only bacteria that possess the cloning vector will live and produce
bacterial colonies)
(c)
Second, one has to make sure that the cloned vector contains an
insertion of DNA (e.g., via phenotypic visualization of the disruption of the lacZ gene)
(d)
Third, one has to confirm either phenotypically or genotypically that
the inserted DNA is indeed the DNA that one is interested in
(e)
Phenotypic identification can involve looking for a specific enzyme
activity or via the presence of a new gene product within a cell of an
appropriate size and/or confirmed by immunological (antibody) reactivity
(f)
Genotypic identification can involve various nucleic-acid probing techniques or the actual sequencing of DNA
(g)
See Figure 20.4, Using a
nucleic acid probe to identify a cloned gene
(a)
For whatever it is worth, even when one has successfully cloned
a piece of foreign DNA that one is interested in, that is often only
a first step of genetic manipulation
(b)
Often one additionally subclones the foreign gene into more-specialized
cloning vectors in order to additionally (e.g., experimentally) manipulate the
gene or its products; often, though, this subcloning is easier than the
original cloning and identification
steps
(c)
[subcloning (Google Search)] [index]
(a)
One type of specialized cloning vector is
an expression vector, a cloning vector designed specifically to express genes
at high levels, under strong experimental control, or both
(b)
[expression vector (Google Search)] [index]
(a)
Because of the presence of introns in
eukaryotic genes, the expression of eukaryotic genes cloned into
bacteria can be problematic even if the eukaryotic DNA control sequences are
replaced by bacterial ones (e.g., by cloning into an expression vector)
(b)
A common way around this is to clone eukaryotic genes from cDNA rather
than from genomic DNA
(c)
cDNA is synthesized from a mature mRNA template (i.e., fully processed
with introns removed) using reverse transcriptase
enzyme
(d)
See Figure 20.5, Making
complementary DNA (cDNA) for a eukaryotic gene
(e)
A “reason to use eukaryotic host cells for expressing a cloned
eukaryotic gene is that many eukaryotic proteins are heavily modified after
translation, often by the addition of lipid or carbohydrate groups. Bacterial cells
cannot perform any of these processing functions, and if the gene product
requiring such processing is from a mammal, even yeast cells will not be able
to modify the protein correctly. The use of host cells from an animal or plant
cell culture may therefore be necessary.”
(f)
[cDNA, complementary DNA (Google Search)] [index]
(17) Polymerase chain reaction (PCR)
(a)
An alternative means of generating large numbers of copies of a
specific piece of DNA (i.e., other than by cloning DNA) is to
employ polymerase chain reaction (PCR)
(b)
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)
(c)
The primers supply the 3’ –OH “primer” necessary for the initiation of
DNA replication
(d)
Polymerization is used to produce double-stranded DNA (i.e., a double
helix) using single-stranded DNA as the template
(e)
Individual single-strands of DNA are typically formed via the unwinding
of a DNA double helix by the application of heat
(f)
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
(g)
See Methods: The Polymerase
Chain Reaction (PCR) found on page 371
(h)
The really neat trick of PCR, however, is to do all of this within 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
(i)
The key step in the development of this technique, therefore, was the
isolation and use of a heat-resistant DNA polyermase (Tac DNA polymerase)
(j)
A key advantage that PCR has
over the conventional cloning of DNA is that PCR may be initiated using only
very small, impure samples of DNA
(k)
[PCR, polymerase chain reaction,
thermocycler (Google)] [index]
(a)
The manipulation of DNA does not end with its cloning
(b)
A number of methods may be employed to analyze the cloned DNA as well
as to compare that DNA with other samples of DNA
(c)
Among these many samples of DNA analysis include:
(ii)
Restriction fragment length
polymorphism (RFLP)
(iii)
In situ hybridization
(iv)
DNA sequencing
(v)
Etc.
(d) &nb