Important words and concepts from Chapter 20, Campbell & Reece, 2002 (1/29/2005):
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(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
(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
(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)
(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
(iv) Gel electrophoresis
(v) Various blotting techniques (Southern blotting, Northern blotting, Western blotting, etc.)
(vi) RFLP analysis
(vii) DNA sequencing
(a) What is molecular biology? Good question
(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
(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)
(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, 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
(e) "For cloning genes or other pieces of DNA, plasmids are first isolated from bacterial cells. FIGURE 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."
(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
(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, Using a restriction enzyme and DNA ligase to make recombinant DNA
(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, 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)
(a) Sticky ends (as well as blunt ends) may be covalently bonded together using the enzyme DNA ligase; see figure to right -->
(b) See Figure, 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)
(a) Once the DNA coming from two different organisms has been ligated together, we can now call it recombinant DNA
(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, 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)
(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, 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."
(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
(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 polymerase (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
(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
(g) See Figure, Using a nucleic acid probe to identify a cloned gene
(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
(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
(a) Once you have a large amount of cloned (or PCRed) DNA, you can tag it either radioactively or fluorescently and then observe where on an intact chromosome that DNA binds
(b) This is useful both for mapping cloned genes to regions on parent chromosome, or for identifying and locating similar genes found among different species
(19) Analysis of cloned DNA (analysis of DNA)
(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:
(iii) In situ hybridization
(iv) DNA sequencing
(d) Some of these methods will be considered below
(a) One of the most useful techniques used for studying macromolecules (particularly proteins and nucleic acids) is gel electrophoresis
(b) Gel electrophoresis is a method of molecule (or complex) separation in which molecules move within a gel-like medium (think Jell-O or, even better, the agar found in a petri dish)
(c) In gel electrophoresis charged molecules are pulled through the gel (typically consisting of the polymer polyacrylamide or of purified agar known as agarose)
(i) Larger or more diffuse molecules move more slowly because they tend to get hung up in the gel matrix
(ii) Molecules with greater charges also move faster because it is an electric voltage that is employed to pull the molecules through the gel matrix
(d) Often to negate charge differences between molecules as well as shape differences (e.g., as when comparing proteins) samples are first mixed with a charged detergent (SDS, a detergent you will typically find in shampoos) that serves to both denature and to even out and increase the amount of negative charge displayed by individual molecules
(e) See Methods: Gel Electrophoresis of Macromolecules
(a) Once you have DNA molecules separated on a gel it is possible to identify molecules that carry specific sequences
(b) This identification is accomplished via the denaturation (separation of individual DNA strands) and then transfer of the DNA found in the gel to a kind of paper that is laid against the gel
(c) The DNA now found on the paper is probed with tagged DNA fragments that, ideally, only bind to specific bands of DNA now found on the paper
(d) The tagged fragments (e.g., radioactively tagged) are then visualized and the location or presence of the DNA on the original gel is inferred from its location or presence on the paper
(e) See Methods: Restriction Fragment Analysis by Southern Blotting
(a) RFLP analysis, along with PCR, was introduced to Americans particularly during the O. J. Simpson trial
(b) The location of restriction sites in a genome occurs fairly randomly, and can differ from person to person essentially as allelic (mutational; nucleotide-sequence) difference between individuals (in general we call differences in nucleotide sequences at specific loci found within populations polymorphisms, in its originally meaning implying differences in the morphologies of individuals making up a population, implying genotypic differences)
(c) 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
(d) The variation between individuals is called restriction fragment length polymorphism
(e) 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)
(f) RFLP analysis these days is employed for just about every population study you can imagine, used in everything from agriculture to forensics to wildlife biology
(g) See Methods: Restriction Fragment Analysis by Southern Blotting
(a) Ultimately genotype consists of sequences of DNA nucleotides (or RNA nucleotides in the case of RNA viruses)
(b) To determine genotype, one must determine the sequence of nucleotides and this is accomplished via a variety of techniques of DNA sequencing
(c) These techniques involve DNA polymerization that is primed by specific, in vitro-synthesized oligonucleotides (short single-stranded DNA polymers) that serve to start the polymerization step at a certain point (essentially at the beginning of the to-be-determined nucleotide sequence)
(d) Typically these oligonucleotides are tagged (e.g., radioactively or, more recently, fluorescently) such that the DNA strand that results from the DNA polymerization may be later visualized
(e) Also required is some means of chain termination that is specific for individual bases plus the separation of DNAs to form single-stranded products
(f) In this manner one ends up with a series of single-stranded DNAs that are tagged at one end for visualization and are of a specific length that is dependent upon where chain termination occurred (i.e., tag + primer + DNA chain/polymer + terminating nucleotide, found together in that order)
(g) These DNAs are then separated via gel electrophoresis such that shorter DNAs (those that are cleaved earlier, i.e., have shorter DNA chains/polymers) migrate farther than longer DNAs (those that are cleaved later, i.e., have longer DNA chains/polymers)
(h) By comparing the migration of DNAs in which chain termination occurred at A, T, G, or C nucleotides (typically one lane in a gel for each terminating nucleotide), one can infer the order that these nucleotides are found in the original DNA molecule (and for those of you who really get what I am saying but at this point are crying, "Foul!" please note that conditions are set up so that chain termination occurs with sufficiently low probability during the sequencing reaction that the polymerization of relatively long DNAs is possible, i.e., rather than each reaction terminating at the first A, T, G, or C found in a sequence)
(i) See Methods: Sequencing of DNA by the Sanger Method found
(24) Vocabulary [index]
(d) Cloning vector
(f) DNA ligase
(g) DNA sequencing
(j) Gene cloning
(r) Recombinant DNA
(w) Restriction site
(z) Sticky ends
Chapter 20, Bio 113 questions:
(#) The application of an electric voltage is employed to separate macromolecules by size, e.g., as when sequencing DNA. This general method of protein or DNA fragment separation is called ___________.
A: Gel electrophoresis
(#) What sort of cloning vehicle would one ligate a piece of DNA into if one were interested in promoting the transcription of that DNA?
A: An expression vector
(#) The ligation together of DNA obtained from two different organisms, e.g., cDNA from a eukaryote and a plasmid obtained from a bacterium, produces what is generally referred to as __________ DNA.
(#) The process of gene cloning typically begins with the _________ and _________ of DNA obtained from a specific organism.
A: Isolation and purification
(#) "Capillary action pulls an alkaline solution upward through the gel and through a sheet of nitrocellulose paper laid on top of it, transferring the DNA to the paper and denaturing it in the process. The single strands of DNA stick to the paper, positioned in bands exactly as on the gel." This is a description of the __________ step of RFLP analysis.
(iv) Hybridization with radioactive probes
(v) Restriction fragment preparation
A: (ii) Blotting
(#) What enzymes are employed to cut genes out of an organism's genome prior to the cloning of these genes?
A: Restriction endonucleases/enzymes
(#) Besides specificity for the replication of specific sequences of DNA, what do the primers employed in the polymerase chain reaction supply and why is there a requirement for two distinctly different primers for any given PCR procedure?
A: They supply 3' --OH groups and one needs two of them because PCR involves the replication of both strands of DNA (the process would not increase DNA concentrations exponentially if only a single primer were supplied)
(#) Beginning at the point where DNA has already been separated via gel electrophoresis, describe the Southern blotting procedure.
A: Southern blotting involves the transfer (blotting) of the DNA to (nitrocellulose) paper followed by the probing of that DNA with tagged DNAs of specific sequence; note that in for this to occur the transferred DNA must be denatured to the single-stranded state, and that the probes, too, must be single stranded (i.e., the entire procedure is based on the propensity for complementary DNAs to hydrogen bond to form double helices)
(#) Describe how/why DNA ligase is employed in gene cloning procedures.
A: There are a variety of possible answers including: DNA ligase is employed to close gaps between adjacent nucleotides in DNA molecules making up one-half of double-helices; DNA ligase is employed to attach DNA pieces to plasmids into which the pieces have been inserted; DNA ligase covalently attaches DNAs that adhere via sticky ends, or even that adhere (transiently) via blunt ends
(#) In gel electrophoresis, contrast the physical characteristics of molecules that migrate rapidly from ones that migrate more slowly. Ignore considerations of charge differences between the molecules.
A: More-slowly migrating molecules tend to be larger and more diffuse than more-rapidly migrating molecules; the reason for this slower migration is that greater size increases the duration of and propensity for interactions between the molecule and the gel matrix
(#) What does RFLP stand for?
A: Restriction Fragment Length Polymorphism
(#) Typically, what type of enzymes does one employ to create DNA "sticky ends"?
(i) DNA polymerase
(iii) Restriction endonuclease
(iv) Reverse transcriptase
(v) RNA polymerase (holoenzyme)
A: (iii) Restriction endonucleases
(#) Cloning vectors consist particularly of what macromolecular substance?
A: (ii) DNA
(#) Complementary DNA consists of a DNA copy of __________, which typically had been isolated from a eukaryotic cell.
A: (ii) mRNA
(#) Given approximately equal charge, what molecular property will lead to slower migration through a gel in gel electrophoresis? Why?
A: Larger molecules because these are more likely to become hung up in the gel matrix
(#) Radioactively (or otherwise) tagged DNA fragments are hybridized to DNA extracted from gels in a process known as Southern __________.
(iv) Restriction fragment
A: (i) Blotting
(#) Which descriptor is most closely associated particularly with the engineering of phenotype?
(ii) DNA technology
(iii) Genetic engineering
(iv) Molecular genetics
(v) Molecular techniques
A: (i) Biotechnology
(#) What is meant by the phrase "gene cloning"?
A: Gene cloning is the growth of fragments of DNA within an organism in which those fragments have been artificially placed
(#) The enzymes employed to cut DNA at specific locations are called __________.
A: Restriction endonucleases; restriction enzymes
(#) Resistance to antibiotics, the disruption of genes (such as the lacZ gene), and phenotypic or genotypic confirmation are all involved in what important (and difficult) step of gene cloning?
A: Identification of a clone
(#) Which is the proper order during PCR reactions?
(i) Heating, unwinding, priming, polymerization
(ii) Priming, heating, polymerization, unwinding
(iii) Priming, unwinding, heating, polymerization
(iv) Priming, unwinding, polymerization, heating
(v) Unwinding, heating, polymerization, priming
A: (i) Heating, unwinding, priming, polymerization
(#) Which is not true of cDNA?
(i) Consists of DNA
(ii) Lacks introns
(iii) Made from genomic DNA
(iv) Made from mature mRNA
(v) Polymerized using reverse transcriptase
A: (iii) Made from genomic DNA
(#) In PCR, primers supply what to Taq polymerase? Please be precise in your answer.
A: 3' --OH
(#) A vector specifically designed to enhance cloned-gene transcription is described as a(n) __________ vector.
(#) What are sticky ends? Please be thorough in your answer, though not necessarily excessively verbose.
A: These are 3' or 5' overhangs that are generated upon restriction endonuclease digestion of DNA
(#) Using traditional DNA sequencing technologies (i.e., the Sanger method) the resulting truncated oligonucleotides are separated employing __________, an otherwise quite common means of DNA separation.
A: Gel electrophoresis
(#) What types of enzymes are required for RFLP analysis?
A: Restriction enzymes
(#) What is a restriction site?
A: The actual nucleotide sequence on a piece of DNA that a restriction enzyme cuts
(#) A cloning vector designed specifically to express genes at high levels is called a(n) __________.
A: Expression vector
(#) Why is it difficult to express cloned eukaryote gene in a bacterium unless one clones those genes as cDNA?
A: The problem is one of introns which are not spontaneously removed from mRNA in prokaryotic systems
(#) In the gel __________ of proteins, to negate charge differences between molecules, samples are first mixed with the detergent, SDS.
(#) True or False, in DNA sequencing, shorter DNAs migrate faster than loner DNAs.