Module 3 Exam

Expression Vector

Cloning vector containing DNA sequences such as a promoter, a ribosome-binding site, and transcription initiation and termination sites that allow DNA fragments inserted into the vector to be transcribed and translated.

Concept

DNA fragments can be inserted into cloning vectors, stable pieces of DNA that will replicate within a cell. A cloning vector must have an origin of replication, one or more unique restriction sites, and selectable markers. An expression vector contains additional sequences that allow a cloned gene to be transcribed and translated. Special cloning vectors have been developed for introducing genes into eukaryotic cells.

Specific DNA Fragments can be amplified

Many of the methods used to manipulate and analyze DNA sequences cannot be carried out on single molecules, requiring instead numerous copies of a specific DNA fragment. A major problem in working at the molecular level is that each gene is a tiny fraction of the total cellular DNA. Because each gene is so rare, it must be isolated and amplified before it can be studied. There are two basic approaches to amplifying a specific DNA fragment: replicating the DNA within cells (in vivo), or replicating the DNA enzymatically outside of cells (in vitro).

DNA Fingerprinting

Technique used to identify individuals by examining their DNA sequences.

Concept

The development of CRISPR-Cas9 technology provides a powerful means of cutting and editing the genome. CRISPR-Cas9 combines a single guide RNA with a nuclease, which together attach to specific DNA sequences and make double-stranded cuts at specific locations. Repair of these cuts by nonhomologous end joining or homologous recombination provides the means to introduce alterations to the genome. This technology has been used in many organisms and cell types, and it has the potential for additional applications, but its use has raised a number of ethical concerns.

Since that discovery, the field has witnessed at least four major transformations, each driven by the development of powerful new techniques that provided important new insights into basic genetic processes.

(1) the development of recombinant DNA technology, which allowed DNA from different sources to be combined; (2) the invention of the polymerase chain reaction, which allowed very small quantities of specific DNA fragments to be quickly amplified; (3) the development of quick and accurate methods of determining DNA sequences; (4) the engineering of CRISPR-Cas systems for accurate and efficient editing of genome sequences.

An effective cloning vector has three important characteristics:

* (1) an origin of replication, which ensures that the vector is replicated within the cell; (2) selectable markers, which enable any cells containing the vector to be selected or identified; and (3) one or more unique restriction sites into which a DNA fragment can be inserted. The restriction sites used for cloning must be unique; if a vector is cut at multiple recognition sites, several pieces of DNA are generated, and getting those pieces back together in the correct order is possible, but extremely difficult.

advantage of cDNA library

* A cDNA library has two additional advantages. First, it is enriched with fragments from actively transcribed genes. Second, introns do not interrupt the cloned sequences; introns would pose a problem when the goal is to produce a eukaryotic protein in bacteria because most bacteria have no means of removing the introns.

Screening DNA Libraries

* A common way to screen DNA libraries is with probes (Figure 19.15). But how is a probe obtained when the gene of interest has not yet been isolated? One option is to use a similar gene from another organism as the probe. * Synthetic probes * Yet another method of screening a DNA library is to look for the protein product of a gene. This method requires that the DNA library be cloned in an expression vector. The clones can be tested for the presence of the protein by using an antibody that recognizes the protein or by using a chemical test for the protein. This method depends on the existence of an antibody or test for the protein produced by the gene of interest. DNA libraries can also be screened using PCR or by sequencing,

LIMITATIONS AND CHALLENGES OF CRISPR-Cas9 EDITING

* A limitation in the use of CRISPR-Cas9 for genome editing is the potential for off-target cleavage. Some mismatches between the sgRNA and the complementary DNA sequence are tolerated by the Cas9 protein, so the system may cleave DNA at sites other than the desired target sequence. Unfortunately, off-target cleavage depends on many factors and varies among cell types, so predicting where and when cleavage will occur has been difficult. Much work has been concentrated on engineering the sgRNA and Cas9 protein to be more selective in pairing and cutting so as to reduce undesirable off-target cuts. For example, geneticists have produced a high-fidelity Cas9 by altering its amino acid sequence so that nonspecific interaction between the Cas protein and the DNA backbone is weakened, thereby forcing the nuclease to depend more on base pairing between the sgRNA and the DNA and increasing its specificity. These changes eliminated nearly all off-target cleavage by the high-fidelity Cas9. * If CRISPR-Cas is applied to multicellular embryos or whole organisms, there is also the potential to create genetic mosaics (see Section 6.2). Genome editing with CRISPR-Cas is not 100% efficient, meaning that DNA in some cells is edited and DNA in other cells is not. It can therefore produce tissues that are mosaics, in which different cells have different DNA sequences. The effects of this mosaicism could vary tremendously, depending on what genes are edited, what tissues are involved, and how efficient the editing is. * Another potential difficulty is getting the CRISPR-Cas components into a cell. This can often be done with cells in culture by transfection, in which cells are bathed in a solution containing DNA or mRNA encoding the Cas protein and the sgRNA and treated to make their membranes more permeable to foreign DNA. While transfection works in cell culture, getting the CRISPR-Cas components into intact organisms is more challenging. Sometimes the sgRNA and Cas protein are directly injected into fertilized egg cells, but most cells are too small for direct injection. Cas9 is a relatively large protein that does not easily pass across the cell membrane. Some other CRISPR-associated nucleases are smaller and easier to get into cells; these smaller nucleases are being exploited for use in delivery.

CRISPR-Cas System

* A molecular tool used for precise editing of DNA that relies on the action of CRISPR RNAs and Cas proteins. * The CRISPR array is transcribed into a long precursor CRISPR RNA (pre-crRNA), which is cleaved into short crRNAs. The crRNAs combine with proteins called CRISPR-associated (Cas) proteins to form effector complexes. The Cas proteins have nuclease activity—the ability to cut DNA. If the same foreign DNA (the protospacer) enters the cell in the future, a CRISPR-Cas complex recognizes and attaches to it. The crRNA in the complex binds to its complementary sequence in the foreign DNA, and the Cas protein cleaves the foreign DNA, rendering it nonfunctional. In this way, CRISPR-Cas serves as an adaptive RNA defense system that remembers and destroys foreign invaders.

GENOME EDITING WITH CRISPR-Cas

* A variety of CRISPR-Cas systems have been found in different bacterial and archaeal species. Geneticists have engineered some of these systems to serve as molecular editing tools. The most widely used system is CRISPR-Cas9, derived from the bacterium Streptococcus pyogenes. This system naturally requires two RNA molecules, crRNA and another RNA molecule termed tracrRNA. These two RNAs pair and then combine with Cas9 to form an effector complex. To facilitate the use of this system in genome editing, researchers have engineered the crRNA and the tracrRNA into a single guide RNA (sgRNA) (Figure 19.3). A 20-nucleotide region of the sgRNA pairs with DNA, although the nucleotides within an 8-12-nucleotide "seed" sequence are most important in pairing. By altering the sequence of the sgRNA, it is possible to direct the action of the effector complex to any specific DNA sequence desired. This relatively long recognition sequence makes CRISPR-Cas9 much more specific than restriction enzymes, meaning that it can be directed to unique sites within the genome.

ADVANTAGES OF CRISPR-Cas9

* An advantage of CRISPR-Cas9 over restriction enzymes is the relatively long nucleotide sequence recognized by the sgRNA, which allows researchers to produce unique cuts within genomic DNA. By changing the sequence of the sgRNA, precise edits can be made almost anywhere in the genome. Modifying the sgRNA to match a particular sequence in the genome is much simpler than trying to alter a DNA-binding protein, such as the proteins in zinc-finger nucleases and TALENS. Another important feature of CRISPR-Cas9 is the ability to use this technology in intact cells; mRNA for the Cas9 protein and specific sgRNAs can be introduced into many types of cells, where they are expressed and carry out their editing function. In a process known as multiplexing, it is possible to introduce several sgRNAs into a cell simultaneously and carry out multiple cuts in a single step. * CRISPR-Cas has great potential for genetic engineering and biotechnology. It can be applied to many different species, including species in which other methods of DNA manipulation have not worked well. It can be used to introduce new DNA sequences into whole animals and humans. For example, it is already being used to induce specific mutations in mice to create genetic models of human diseases, which can then serve as powerful research tools for the study of those diseases. CRISPR-Cas is also being developed as a tool for correcting genetic defects (gene therapy) and has the potential for treating infectious diseases by eliminating viral DNA from human cells. It will enable genetic modification of crops and domestic animals, in which it can be used to create very specific genetic alterations that benefit yield and produce characteristics that improve cultivation. * Researchers are already developing modifications of CRISPR-Cas systems to provide additional functions. For example, the Cas9 protein can be modified so that it makes single-stranded cuts in DNA, which are more likely to be repaired by homologous recombination and other precise DNA-repair mechanisms. Nucleases of other CRISPR systems are being used for different types of cutting. For example, a Cas protein called Cpf1, which is used in some CRISPR systems, makes staggered cuts that produce complementary sticky ends like those produced by some restriction enzymes. The production of complementary sticky ends makes repair more accurate and enables the insertion of desired sequences with complementary sticky ends. * When a PAM for Cas9 is not available near a site where cleavage is desired, Cas proteins from different species that recognize different PAMs can be used. There is the potential to use a modified CRISPR-Cas system (with its cleavage function inactivated) as a general RNA-guided device for other functions, such as targeted transcriptional activation and transcriptional silencing. Transcriptional activator proteins or chromatin-remodeling proteins can be tethered to Cas to bring about transcription at specific sites in the genome.

Zinc-fingered nucleases (ZFNs)

* An engineered nuclease consisting of an array of zinc-finger domains attached to a restriction enzyme. * which use a DNA-binding domain called a zinc finger (see Section 16.1) attached to a restriction enzyme, most often FokI. In these nucleases, several zinc-finger domains are combined in an array; the most commonly used ZFNs have three of these domains, which together recognize a 9-bp DNA sequence. To make double-stranded cuts in DNA, ZFNs are used in pairs, which increases their specificity to 18 or more base pairs—enough to ensure that most genomes are cut only once.

Transcription activator-like effector nuclease (TALEN)

* An engineered nuclease in which a protein of a type that normally binds to promoters is attached to a restriction enzyme. * in which a protein that normally binds to sequences in promoters is attached to the FokI restriction enzyme. The binding protein consists of a series of repeats whose amino acid sequence determines the DNA base sequence that it recognizes.

CRIPSR-Cas Genome Editing

* Another molecular tool for precisely cutting DNA is the CRISPR-Cas system that has been developed in recent years. This technique has revolutionized the field of genetics, providing a powerful way of editing the genome that has now been applied to DNA sequences in bacteria, yeast, nematodes, plants, fruit flies, zebrafish, mice, rats, monkeys, humans, and many more organisms.

The CRISPR-Cas systems occur naturally in _____.

* Bacteria

SHOTGUN CLONING

* Before the development of low-cost sequencing methods, genes were often located by first creating libraries of DNA sequences and then screening those libraries for genes of interest. This approach—to clone first and search later—is called shotgun cloning because it is like hunting with a shotgun: the pellets spray widely in the general direction of the target, with a good chance that one or more of the pellets will hit it. In shotgun cloning, a researcher first clones a large number of DNA fragments, knowing that one or more contains the DNA of interest, and then searches for the fragment of interest among the clones.

RISPR-Cas IMMUNITY IN BACTERIA AND ARCHAEA

* CRISPR-Cas systems occur naturally in bacteria and archaea and are used to protect these organisms against bacteriophages, plasmids, and other invading DNA elements (see Chapters 9 and 14). CRISPR RNAs (crRNAs) are encoded by DNA sequences called Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR). A CRISPR array consists of a series of such palindromic sequences (sequences that read the same forward and backward on two complementary DNA strands) separated by unique spacers, which consist of sequences derived from bacteriophages or foreign plasmids (see Figure 14.23a). When bacteriophage or plasmid DNA enters a prokaryotic cell, proteins cut up the foreign DNA and insert bits of it into a CRISPR array, which then serves as a memory of the invader. The DNA sequences in bacteriophage or plasmid DNA that match the spacer elements in the CRISPR array are referred to as protospacers.

Yeast Artificial Chromosome (YAC)

* Cloning vector consisting of a DNA molecule with a yeast origin of replication, a pair of telomeres, and a centromere. YACs can carry very large pieces of DNA (as large as several hundred thousand base pairs) and replicate and segregate as yeast chromosomes do. * is a DNA molecule that has a yeast origin of replication, a pair of telomeres, and a centromere. These features ensure that YACs are stable and that they replicate and segregate in the same way as yeast chromosomes. YACs are particularly useful because they can carry DNA fragments as large as 600 kb, and some special YACs can carry inserts of more than 1000 kb. YACs have been modified so that they can be used in eukaryotic organisms other than yeast.

Concept

* DNA fingerprinting detects genetic differences among individuals by analyzing highly variable regions of chromosomes.

Concept

* DNA fragments can be separated, and their sizes determined, with the use of gel electrophoresis. The fragments can be viewed by using a dye that is specific for nucleic acids or by labeling the fragments with a chemical tag.

Gene Cloning

* Despite the widespread use of PCR, some DNA sequences are still amplified by gene cloning. In addition, gene cloning provides a powerful means of altering and manipulating DNA sequences. It is often used to alter cells so that they have desired properties or produce substances of commercial value.

Restriction Enzymes (Endonucleases)

* Enzyme that recognizes particular base sequences in DNA and makes double-stranded cuts nearby; also called a restriction endonuclease. * The sequences recognized by restriction enzymes are usually from 4 to 8 bp long; most of these enzymes recognize a sequence of 4 or 6 bp. Most recognition sequences are palindromic—sequences that read the same (5′ to 3′) on the two complementary DNA strands.

3rd-Generation Sequencing

* Even more advanced and rapid sequencing methods, typically called third-generation sequencing technologies, are currently under development. Nanopore sequencing, for example, can determine the sequence of individual molecules of DNA. In this method, a single strand of DNA is passed through a tiny hole—a nanopore—in a membrane. As the molecule passes, one nucleotide at a time, through the nanopore, it disrupts an electrical current in the membrane, and the nature of the disruption is affected by the shape of the nucleotide passing through the nanopore. Each of the four bases of DNA causes a characteristic disruption, so the sequence of DNA can be read by analyzing the membrane current as the strand passes, one nucleotide at a time, through the nanopore. Hundreds of thousands of nanopores can be created on a single chip, so that many DNA fragments can be read simultaneously.

Disadvantages and Advantages of Gene Cloning

* For many years, all amplification of DNA was done by gene cloning. A major disadvantage of gene cloning is the time required: the process of inserting the DNA into bacteria, selecting and growing the bacterial cells that have incorporated it, and isolating the amplified DNA usually requires several days. Gene cloning is also relatively labor-intensive, requiring a number of steps that are difficult to automate. An advantage is that, because it uses the cell's high-fidelity replication machinery, gene cloning typically copies DNA with great accuracy.

What is a major challenge faced by a molecular geneticist?

* Genes only make up a small percentage of the total DNA in a cell.

Which next-generation sequencing technique is most similar to the Sanger sequencing method?

* Illumina

Vivo

* In the in vivo approach, a DNA fragment is inserted into a bacterial cell and the cell is allowed to replicate the DNA. Each time the cell divides, one or more copies of the DNA fragment are passed on to each daughter cell. Most bacterial cells divide rapidly, so within a short time (usually a few days), a large number of genetically identical cells are produced, each carrying one or more copies of the DNA fragment. The cells are then lysed to release their DNA, and the desired fragment is isolated from the rest of the bacterial DNA. This procedure is termed gene cloning because identical copies (clones) of the original piece of DNA are replicated within bacterial cells.

Gene Cloning

* Insertion of DNA fragments into bacteria in such a way that the fragments will be stable and will be copied by the bacteria. * This procedure is termed gene cloning because identical copies (clones) of the original piece of DNA are replicated within bacterial cells.

Probe

* Known sequence of DNA or RNA that is complementary to a sequence of interest and will pair with it; used to find specific DNA sequences.

Concept

* Labeled probes, which are sequences of RNA or DNA that are complementary to the sequence of interest, can be used to locate individual genes or sequences among DNA fragments separated by electrophoresis.

Ti plasmid

* Large plasmid isolated from the bacterium Agrobacterium tumefaciens and used to transfer genes to plant cells.

Polymerase Chain Reaction (PCR)

* Method of enzymatically amplifying DNA fragments. * a technique first developed in 1983 by Kary Mullis.

Chromosomal Walking

* Method of locating a gene by using partly overlapping genomic clones to move in steps from a previously cloned, linked gene to the gene of interest. * In chromosome walking, neighboring genes are used to locate a gene of interest.

Positional Cloning

* Method that allows for the isolation and identification of a gene by examining the cosegregation of a phenotype with previously mapped genetic markers. * Isolation of genes on the basis of their position on a gene map. * For many genes with important functions, no associated protein product has yet been identified. The biochemical bases of many human genetic diseases, for example, are still unknown. How can these genes be isolated? One approach is to first determine the general location of the gene on a chromosome by using recombination frequencies derived from crosses or pedigrees (see Chapter 7). After the chromosomal region where the gene is found has been pinpointed, genes in that region can be cloned and identified. Then other techniques can be used to determine which of the "candidate" genes might be the one that causes the disease. This approach—isolation of genes on the basis of their position on a gene map—is called positional cloning. * In the first step of positional cloning, geneticists use mapping studies (see Chapter 7) to establish linkage between molecular markers and a phenotype of interest, such as a human disease or a desirable physical trait in a plant or animal. Demonstration of linkage between the phenotype and one or more molecular markers tells us which chromosome carries the locus that codes for the phenotype and its general location on that chromosome. The next step is to narrow down the location of the gene by using additional molecular markers clustered in the chromosomal region where the gene resides. Clones that cover that region can then be isolated from a genomic library. With the use of a technique called chromosome walking (Figure 19.17), it is possible to progress from clones of molecular markers to linked clones, one of which might contain the gene of interest. The basis of chromosome walking is the fact that a genomic library consists of a set of overlapping DNA fragments (see Figure 19.14). We start with a cloned gene marker that is close to the new gene of interest so that the "walk" will be as short as possible. One end of the clone of a neighboring marker (clone A in Figure 19.17) is used to make a complementary probe. This probe is used to screen the genomic library to find a second clone (clone B) that overlaps with the first and extends in the direction of the gene of interest. This second clone is isolated and purified, and a probe is prepared from its end. The second probe is used to screen the library for a third clone (clone C) that overlaps with the second. In this way, one can systematically "walk" toward the gene of interest, one clone at a time. A related technique, called chromosome jumping, allows one to move from more distantly linked markers to clones that contain a sequence of interest.

IN SITU HYBRIDIZATION

* Method used to determine the chromosomal location of a gene or other specific DNA fragment or the tissue distribution of an mRNA by using a labeled probe that is complementary to the sequence of interest. * DNA probes can be used to determine the chromosomal location of a gene in a technique * The name of this technique is derived from the fact that DNA (or RNA) is visualized while it is in the cell (in situ). It requires that the cells be fixed and the chromosomes be spread on a microscope slide and denatured. A labeled probe is then applied to the slide, just as it can be applied to a gel. In fluorescence in situ hybridization (FISH), the probes carry attached fluorescent dyes that can be seen directly with the microscope (Figure 19.16a). Several probes attached to different colored dyes can be used simultaneously to investigate different sequences or chromosomes. FISH has been used to identify the chromosomal location of human genes. In situ hybridization can also be used to determine the distribution of specific mRNA molecules in tissues, serving as a source of insight into how gene expression differs among cell types (Figure 19.16b). A labeled DNA probe complementary to a specific mRNA molecule is added to tissue, and the location of the bound probe is determined by visualizing the radioactive or fluorescent label. Determining where a gene is expressed often helps define its function. For example, finding that a gene is highly expressed only in brain tissue might suggest that the gene has a role in neural function.

Working at the Molecular Level

* Molecular genetic analyses require special techniques because individual genes make up a tiny fraction of the cellular DNA and cannot be seen.

Sanger vs Next Generation

* Most next-generation sequencing techniques read shorter DNA fragments than the Sanger sequencing method can, but because hundreds of thousands or millions of fragments are sequenced simultaneously, these methods are much faster than traditional Sanger sequencing technology. But these techniques only determine the sequences of short DNA fragments; they do not, by themselves, allow the sequences of these fragments to be reassembled into the sequence of the entire original piece of DNA.

Concept

* New next- and third-generation sequencing methods can sequence many DNA fragments simultaneously, providing a much faster and less expensive determination of a DNA base sequence than does the Sanger sequencing method.

Blunts Ends

* Not all restriction enzymes produce staggered cuts and sticky ends (see Figure 19.2a). PvuII cuts in the middle of its recognition sequence, and the cuts on the two strands are directly opposite each other, producing blunt-ended fragments that must be joined together in other ways: * The sequences recognized by a restriction enzyme are located randomly within the genome. Accordingly, there is a relation between the length of the recognition sequence and the number of times it is present in a genome: there will be fewer longer recognition sequences than shorter ones because the probability of the occurrence of a particular sequence consisting of, say, six specific bases is less than the probability of the occurrence of a particular sequence of four specific bases. Therefore, restriction enzymes that recognize longer sequences will cut a given piece of DNA into fewer and longer fragments than will restriction enzymes that recognize shorter sequences.

19.2 Molecular Techniques Are Used to Cut and Visualize DNA Sequences

* Often a first step in the molecular analysis of a DNA segment or gene is to isolate it from the remainder of the DNA so that further analyses can be carried out. In the sections that follow, we examine three groups of molecular techniques that are used to cut DNA segments: restriction enzymes, engineered nucleases, and CRISPR-Cas systems.

Transformation

* Once a DNA fragment of interest has been placed inside a plasmid, the plasmid must be introduced into bacterial cells. This task is usually accomplished by transformation, the mechanism by which bacterial cells take up DNA from the external environment (see Section 9.3). Some types of cells undergo transformation naturally; others must be treated chemically or physically before they will undergo transformation. Inside the cells, the plasmids replicate and multiply as the cells themselves multiply.

REPAIR OF BREAKS PRODUCED BY CRISPR-Cas9

* Once the DNA of a cell has been cleaved by CRISPR-Cas9, the cell immediately uses its DNA-repair mechanism to try to repair the break. This feature provides a mechanism for editing the target sequence. There are two major pathways by which double-strand breaks are repaired within cells (see Section 18.5). One, called nonhomologous end joining, joins together the two ends of DNA without using any template. This process tends to introduce small insertions and deletions when the two ends are joined, a side effect that allows geneticists to disable a gene. The CRISPR-Cas9 system can be targeted to a specific gene; when that gene is cleaved by Cas9 and then repaired by nonhomologous end joining, the introduction of insertions or deletions at the break site often produces frameshift mutations that disrupt the coding sequence and disable the gene (see Figure 19.3). * The other mechanism used by cells to repair double-strand breaks is homologous recombination (see Section 18.5). This mechanism functions when a DNA template is provided for repairing the break. A researcher can provide a donor piece of double-stranded DNA that has ends complementary to the sequences at the ends of the break made by Cas9; homologous recombination may insert the donor DNA sequence into the break. In this way, researchers can selectively insert a desired sequence into a genome (see Figure 19.3). Unfortunately, homologous recombination is not highly efficient, and often the two ends are connected without insertion of the donor DNA.

Concerns about CRISPR-Cas9 Editing

* One concern about CRISPR-Cas9 technology is that its ease of use and potential to alter almost any DNA sequence might mean that it could be used to genetically modify humans in ethically questionable ways. The CRISPR-Cas system has already been used to carry out genome editing in human embryos, although these embryos were not capable of completing development and producing live-born humans. Nevertheless, it is clear that the technology can be used for genetically modifying humans, and this observation has raised considerable debate. Most ethical concern focuses on germ-line editing (in which genes in reproductive cells are altered) because the edited genes will be passed on to future generations, affecting the future gene pool of the species. There are also safety concerns about applying CRISPR-Cas to humans. Off-target cuts could induce mutations that might lead to cancer or other medical problems. Some researchers have pointed to the potential dangers of using CRISPR-Cas to edit animals and plants that are released into the wild. In early 2015, a group of leading scientists and ethicists met in Napa, California, to discuss the scientific, legal, and ethical implications of genome editing using CRISPR-Cas technology. This group strongly discouraged all attempts at human germ-line modification until the implications of CRISPR-Cas editing could be discussed more widely by scientific and governmental organizations. The group encouraged transparent research, educational forums on the new technology, and the convening of

Concept

* One method of finding a gene is to create and screen a DNA library. A genomic library is created by cutting genomic DNA into overlapping fragments and cloning each fragment in a separate bacterial cell. A cDNA library is created from mRNA that is converted into cDNA and cloned in bacteria.

In addition to amplifying DNA, (PCR)

* PCR can be used to amplify sequences corresponding to RNA. This amplification is accomplished by first converting RNA into complementary DNA (cDNA) with reverse transcriptase. The cDNA can then be amplified by the usual PCR. This technique is referred to as reverse-transcription PCR.

Plasmid Vectors

* Plasmids, circular DNA molecules that exist naturally in bacteria (see Chapter 9), are commonly used as vectors for cloning DNA fragments in bacteria. They contain origins of replication and are therefore able to replicate independently of the bacterial chromosome. The plasmids typically used in cloning have been artificially constructed from larger, naturally occurring bacterial plasmids and have multiple unique restriction sites and selectable markers as well as an origin of replication * The easiest method for inserting a DNA sequence into a plasmid vector is to cut the foreign DNA (containing the DNA fragment of interest) and the plasmid with the same restriction enzyme (Figure 19.8). If the restriction enzyme makes staggered cuts in the DNA, complementary sticky ends are produced on the foreign DNA and the plasmid. When DNA and plasmids are then mixed together, some of the foreign DNA fragments will pair with the cut ends of the plasmids. DNA ligase is used to seal the nicks in the sugar-phosphate backbone, creating a recombinant plasmid that contains the foreign DNA fragment.

Concepts

* Positional cloning allows researchers to isolate a gene without having knowledge of the biochemical basis of its phenotype. Linkage studies are used to map the locus producing a phenotype of interest to a particular chromosome region. Chromosome walking and jumping can be used to progress from molecular markers to clones containing sequences that cover the chromosome region. Candidate genes within the region are then evaluated to determine whether they encode the phenotype of interest.

Southern Blotting (DNA)

* Process by which DNA is transferred from a gel to a solid support such as a nitrocellulose or nylon filter.

Northern Blotting (RNA)

* Process by which RNA is transferred from a gel to a solid support such as a nitrocellulose or nylon filter

Western Blotting.

* Process by which protein is transferred from a gel to a solid support such as a nitrocellulose or nylon filter.

DNA Sequencing

* Process of determining the sequence of bases along a DNA molecule. * a powerful method for analyzing DNA

Whats necessary for PCR?

* Replication by PCR requires a DNA template (the fragment of interest, or target DNA) from which a new DNA strand can be copied, and a pair of single-stranded primers, each with a 3′OH group to which new nucleotides can be added. The primers used in PCR are short fragments of DNA, typically 17-25 nucleotides long, that are complementary to known sequences on the template. To carry out PCR, researchers begin with a solution that includes the target DNA, DNA polymerase, all four deoxyribonucleoside triphosphates (dNTPs—the substrates for DNA polymerase), primers, and magnesium ions and other salts that are necessary for the reaction to proceed.

Next-Generation Sequencing Technologies

* Sequencing methods, such as pyrosequencing, that are capable of simultaneously determining the sequences of many DNA fragments; these technologies are much faster and less expensive than the Sanger dideoxy sequencing method.

Recombinant DNA Technology

* Set of molecular techniques for locating, isolating, altering, combining, and studying DNA segments; also commonly called genetic engineering. * Techniques for accurately and efficiently cleaving DNA helped to usher in the first major revolution in molecular genetics: the development of recombinant DNA technology. In 1973, a group of scientists produced the first organisms with recombinant DNA molecules. The group, led by Stanley Cohen, at Stanford University, and Herbert Boyer, at the University of California, San Francisco School of Medicine, inserted a piece of DNA from one plasmid into another, creating an entirely new, recombinant DNA molecule. They then introduced the recombinant plasmid into E. coli cells. These experiments ushered in one of the most momentous revolutions in the history of science.

pyrosequencing

* Several other forms of next-generation sequencing have been developed. One type, called pyrosequencing, is based on DNA synthesis: nucleotides are added one at a time in the order specified by template DNA and the addition of a particular nucleotide is detected with a flash of light, which is generated as the nucleotide is added. To carry out pyrosequencing, DNA to be sequenced is first fragmented (Figure 19.24a). * An adaptor, consisting of a short string of nucleotides, is added to each fragment. The adaptor provides a known sequence to prime a PCR reaction. The DNA fragments are then made single stranded. In one version of pyrosequencing, each fragment is then attached to a separate bead and surrounded by a droplet of solution containing the reagents for PCR (Figure 19.24b). The bead is used to hold the DNA and, later, to deposit it on a plate for the sequencing reaction, as we'll see shortly. Within the droplet, the fragment is amplified by PCR, and the copies of the fragment remain attached to the bead. After amplification, the beads are mixed with DNA polymerase and are deposited on a plate containing more than a million wells (microscopic holes). Each bead is deposited into a different well.

Cohesive Ends

* Short, single-stranded overhanging end on a DNA molecule produced when the DNA is cut by certain restriction enzymes; also called a sticky end. Cohesive ends are complementary and can spontaneously pair to rejoin DNA fragments that have been cut with the same restriction enzyme. * Such ends are called cohesive ends, or sticky ends, because they are complementary to each other and can spontaneously pair to connect the fragments. Thus, DNA fragments with sticky ends can be "glued" together: any two such fragments cleaved by the same enzyme will have complementary ends and will pair (Figure 19.2). When their cohesive ends have paired, the two DNA fragments can be joined together permanently by DNA ligase, which seals nicks between the sugar-phosphate groups of the fragments.

Linkers

* Small synthetic DNA fragment that contains one or more restriction sites; can be attached to the ends of any piece of DNA and used to insert it into a plasmid vector.

Seperating and Viewing DNA Fragments

* Small wells are made at one end of the gel, solutions of DNA fragments are placed in the wells (Figure 19.4a), and an electrical current is passed through the gel. Because the phosphate group on each DNA nucleotide carries a negative charge, the DNA fragments migrate toward the positive end of the gel. During this migration, the porous gel acts as a sieve, separating the DNA fragments by size. Small DNA fragments migrate more rapidly than do large ones, so, over 1-2 hours, the fragments separate on the basis of their size. Typically, DNA fragments of known length (size standards) are placed in one of the wells. By comparing the migration distance of the unknown fragments with the distance traveled by the size standards, a researcher can determine the approximate size of the unknown fragments (Figure 19.4b). * The DNA fragments are still too small to see, so the problem of visualizing them must be addressed. Visualization can be accomplished in several ways. The simplest procedure is to stain the gel with a dye specific for nucleic acids, such as ethidium bromide, which wedges itself tightly (intercalates) between the bases of DNA and fluoresces when exposed to UV light, producing brilliant bands on the gel (Figure 19.4c). Alternatively, DNA fragments can be visualized by adding a label to the DNA before it is placed in the gel. For example, chemical labels can be detected by adding antibodies or other substances that carry a dye and will attach to the relevant DNA, which can then be visualized directly.

Gene cloning more than replicating the gene

* Sometimes the goal in gene cloning is not just to replicate the gene, but also to produce the protein it encodes. To ensure transcription and translation, a foreign gene is usually inserted into an expression vector, which, in addition to the usual origin of replication, restriction sites, and selectable markers, contains sequences required for transcription and translation in bacterial cells * To ensure transcription and translation, a foreign gene may be inserted into an expression vector

ddNTPs

* Special substrate for DNA synthesis used in the Sanger dideoxy sequencing method; identical with dNTP (the usual substrate for DNA synthesis) except that it lacks a 3′-OH group. The incorporation of a ddNTP into DNA terminates DNA synthesis.

Cloning Vector

* Stable, replicating DNA molecule to which a foreign DNA fragment can be attached for transfer to a host cell.

gel electrophoresis

* Technique for separating charged molecules (such as proteins or nucleic acids) on the basis of molecular size or charge, or both.

Reverse-Transcription PCR

* Technique that amplifies sequences corresponding to RNA; reverse transcriptase is used to convert RNA into complementary DNA, which can then be amplified by the usual polymerase chain reaction.

Concept

* Techniques of molecular genetics are used to locate, analyze, alter, sequence, study, and recombine DNA sequences. These techniques are used to probe the structure and function of genes, address questions in many areas of biology, create commercial products, and diagnose and treat diseases. Four key innovations that have revolutionized genetics include recombinant DNA technology, the polymerase chain reaction, DNA sequencing technology, and genome editing methods.

How are microsatellites detected?

* The STRs are typically detected with PCR, using primers flanking the microsatellite repeats so that a DNA fragment containing the repeated sequences is amplified (Figure 19.25). The length of the amplified segment depends on the number of repeats; DNA from a person with more repeats will produce a longer amplified segment than will DNA from a person with fewer repeats.

In the dideoxy-sequencing reaction, what terminates DNA synthesis at a particular base?

* The absence of a 3′-OH group on the ddNTP prevents the addition of another nucleotide.

A geneticist inserts a DNA fragment into a restriction site within the lacZ gene of a plasmid that also has an ampicillin-resistance gene. The geneticist then transforms lacZ− bacterial cells with the plasmids and plates the bacteria on a medium containing ampicillin. Which cells will have copies of the recombinant plasmid?

* The cells that grow white, because they do not produce B-galactidose * Correct! The cells that grow and remain white have a plasmid in which the inserted DNA fragment has disrupted the functional lacZ gene.

Dideoxy Sequencing

* The dideoxy (or Sanger) method of DNA sequencing is based on replication. The fragment to be sequenced is used as a template to make a series of new DNA molecules. In the process, replication is sometimes, but not always, terminated when a specific base is encountered, producing DNA strands of different lengths, each of which ends in a known base. The method relies on the use of a special substrate for DNA synthesis. Normally, DNA is synthesized from deoxyribonucleoside triphosphates (dNTPs), which have an OH group on the 3′-carbon atom (Figure 19.21a). In the Sanger method, special nucleotides, called dideoxyribonucleoside triphosphates (ddNTPs; Figure 19.21b), are also used as substrates. The ddNTPs are identical with dNTPs, except that they lack a 3′-OH group. In the course of DNA synthesis, ddNTPs are incorporated into a growing DNA strand. However, after a ddNTP has been incorporated into the DNA strand, no more nucleotides can be added because there is no 3′-OH group to form a phosphodiester bond with an incoming nucleotide. Thus, ddNTPs terminate DNA synthesis.

Concept

* The dideoxy-sequencing method uses ddNTPs, which terminate DNA synthesis at specific bases.

Disadvantage of cDNA library

* The disadvantage of a cDNA library is that it contains only sequences that are present in mature mRNA.Sometimes, researchers are interested in sequences that are not transcribed, such as those in promoters and enhancers, and these sequences will not be present in a cDNA library. Furthermore, a cDNA library contains only those genes expressed in the tissue from which the RNA was isolated, and the frequency of a particular DNA sequence in a cDNA library depends on the abundance of the corresponding mRNA in that tissue. So, if a particular gene is not expressed, or is expressed at low frequency, in a particular tissue, it may be absent from a cDNA library prepared from that tissue. In contrast, almost all genes are present at the same frequency in a genomic library.

How are candidate genes that are identified by positional cloning evaluated to determine whether they encode the phenotype of interest?

* The expression pattern of the gene—where and when it is transcribed—can often provide clues about its function; for example, genes for neurological disease would probably be expressed in the brain. Geneticists often look in the coding regions of candidate genes for mutations among individuals with the phenotype of interest.

Two key innovations facilitated the use of PCR in the laboratory.

* The first was the discovery of a DNA polymerase that is stable at the high temperatures used in step 1. The DNA polymerase from E. coli that was originally used in PCR denatures at 90°C, so fresh enzyme had to be added to the reaction mixture in each cycle, which slowed the process considerably. This obstacle was overcome when DNA polymerase was isolated from the bacterium Thermus aquaticus, which lives in the boiling springs of Yellowstone National Park. This enzyme, dubbed Taq polymerase, is remarkably stable at high temperatures and is not denatured in the strand-separation step; it can be added to the reaction mixture at the beginning of the PCR process and will continue to function through many cycles. * The second key innovation was the development of automated thermal cyclers: machines that bring about the rapid temperature changes necessary for the different steps of PCR. Originally, tubes containing reaction mixtures were moved by hand among water baths set at the different temperatures required for the three steps of each cycle. In automated thermal cyclers, the reaction tubes are placed in a metal block whose temperature changes rapidly according to a computer program.

ZFNs and TALENs

* The key to both ZFNs and TALENs is the ability to alter the amino acid sequence of the binding proteins in these nucleases in such a way that they bind to a specific target DNA sequence. For ZFNs, there is no simple correspondence between the amino acid sequence of the binding protein and the DNA sequence to which it binds, so each ZFN must be custom-designed for a particular sequence. For TALENs, a more straightforward relation exists between the amino acid sequence of the binding protein and the base sequence of the target DNA, but making specific proteins for individual DNA sequences is laborious and costly.

Concept

* The polymerase chain reaction is an enzymatic, in vitro method for rapidly amplifying DNA. In this process, DNA is heated to separate the two strands, short primers attach to the target DNA, and DNA polymerase synthesizes new DNA strands from the primers. Each cycle of PCR doubles the amount of DNA. PCR has a number of important applications in molecular biology.

In the polymerase chain reaction (PCR), the solution is first heated to 90°C, then cooled to 30-65°C, and then heated to 60-70°C. What happens when the solution is cooled to 30-65°C?

* The primers anneal. * Correct! The primers anneal at 30-65°C.

Typically, DNA repair mechanisms that introduce small insertions and deletions, like nonhomologous end joining, are not ideal for use by researchers because these mutations could harm the gene. Why would this outcome be desired when using the CRISPR-Cas9 system?

* The researchers are trying to disrupt genes to study functionality, thus mutations are one way to do this.

Vitro

* The second, in vitro, approach is to amplify DNA enzymatically in a test tube outside of cells. This amplification is done with the polymerase chain reaction (PCR), a technique first developed in 1983 by Kary Mullis. The basis of PCR is DNA replication catalyzed by a DNA polymerase. Because a DNA molecule consists of two nucleotide strands, each of which can serve as a template, the amount of DNA doubles with each replication event. PCR allows DNA fragments to be amplified a billionfold within just a few hours, and it can be used with extremely small amounts of original DNA, even a single molecule. The polymerase chain reaction revolutionized molecular biology and is now one of the most widely used molecular techniques.

In the polymerase chain reaction (PCR), the solution is first heated to 90°C. What happens in this first step of the reaction

* The two strands of DNA separate. * Correct. The two DNA strands separate when DNA is heated to 90°C.

Molecular Techniques Can Be Used to Find Genes of Interest

* To analyze a gene or to transfer it to another organism, the gene must be located and isolated. Today, most genes are located by sequencing a genome (see Chapter 20) and determining the locations of genes from the sequence. Before the development of low-cost sequencing methods, genes were often located by first creating libraries of DNA sequences and then screening those libraries for genes of interest. This approach—to clone first and search later—is called shotgun cloning because it is like hunting with a shotgun: the pellets spray widely in the general direction of the target, with a good chance that one or more of the pellets will hit it. In shotgun cloning, a researcher first clones a large number of DNA fragments, knowing that one or more contains the DNA of interest, and then searches for the fragment of interest among the clones.

Creating a cDNA Library

* To create a cDNA library, messenger RNA is first extracted from cells and separated from other types of cellular RNA (tRNA, rRNA, snRNA, etc.). The mRNA molecules are then copied into cDNA using reverse transcriptase, an enzyme isolated from retroviruses (see Section 9.4) that synthesizes single-stranded complementary DNA using RNA as a template. The resulting RNA-DNA hybrid molecule is finally converted into a double-stranded cDNA molecule by DNA polymerase.

Creating a genomic Library

* To create a genomic library, cells are collected and lysed, which causes them to release their DNA and other cellular contents into an aqueous solution, and the DNA is extracted from the solution. After the DNA has been isolated, it is incubated with a restriction enzyme for a limited amount of time so that only some of the restriction sites in each DNA molecule are cut (a partial digestion). Because the cutting of sites is random, different DNA molecules will be cut in different places, and a set of overlapping fragments will be produced (Figure 19.14). The fragments are then joined to vectors and transferred to bacteria. A few of the clones contain the entire gene of interest (if the gene is not too large), and a few contain parts of the gene, but most contain fragments that have no part of the gene of interest. A genomic library must contain a large number of clones to ensure that all DNA sequences in the genome are represented in the library. A library of the human genome formed by using cosmids, each carrying a random DNA fragment from 35,000 to 44,000 bp long, would require about 350,000 cosmid clones to provide a 99% chance that every sequence is included in the library.

Engineered Nucleases

* To overcome this problem of numerous cuts, geneticists have sought nucleases that recognize longer DNA sequences. In recent years, geneticists have designed complex enzymes, termed engineered nucleases, that are capable of making unique double-stranded cuts in DNA at predetermined sequences. Engineered nucleases consist of the part of a restriction enzyme that cleaves DNA nonspecifically, coupled with another protein that recognizes and binds to a specific DNA sequence; the particular sequence to which the protein binds is determined by the protein's amino acid sequence. By altering the amino acid sequence of the binding protein, geneticists can custom-design a nuclease to bind to and cut any particular DNA sequence. * Protein consisting of part of a restriction enzyme, which cleaves DNA, combined with another protein that recognizes and binds to a specific DNA sequence; capable of making double-stranded cuts to the DNA at a predetermined DNA sequence. Engineered nucleases can be custom designed to bind to and cut any particular DNA sequence..

LIMITATIONS OF PCR

* Today, the polymerase chain reaction is often used in place of gene cloning, but it has several limitations. First, the use of PCR requires prior knowledge of at least part of the sequence of the target DNA so that the primers can be constructed. Second, the capacity of PCR to amplify extremely small amounts of DNA makes contamination a significant problem. Minute amounts of DNA from the skin of laboratory workers or small particles in the air can enter a reaction tube and be amplified along with the target DNA. Careful laboratory technique and the use of controls are necessary to circumvent this problem. * A third limitation of PCR is accuracy. Unlike other DNA polymerases, Taq polymerase does not have the capacity to proofread (see pp. 353-354 in Chapter 12), and under standard PCR conditions, it incorporates an incorrect nucleotide about once every 20,000 bp. New heat-stable DNA polymerases with proofreading capacity have been isolated that give more accurate PCR results. * A fourth limitation of PCR is that the size of the fragments that can be amplified by standard Taq polymerase is usually less than 2000 bp. By using a combination of Taq polymerase and a DNA polymerase with proofreading capacity, and by modifying the reaction conditions, investigators have been successful in extending PCR amplification to larger fragments, but even these larger fragments are limited in length to 50,000 bp.

Concept

* Type II restriction enzymes cut DNA at specific base sequences that are palindromic. Some restriction enzymes make staggered cuts, producing DNA fragments with cohesive ends; others cut both strands straight across, producing blunt-ended fragments. There are fewer long recognition sequences in DNA than short recognition sequences.

Biotechnology

* Use of biological processes, particularly molecular genetics and recombinant DNA technology, to produce products of commercial value. * The biotechnology industry has grown up around the use of these techniques to develop new products. In medicine, molecular genetic analysis is being used to probe the nature of cancer, diagnose genetic and infectious diseases, produce drugs, and treat hereditary disorders.

How can a probe for a gene be obtained when the gene has not yet been isolated or sequenced?

* Use the sequence of a similar gene from another organism * Determine the nucleotide sequence of the probe from the amino acid sequence of the protein encoded by the gene

Microsatellite

* Very short DNA sequence repeated in tandem; also called a short tandem repeat.

What would you observe in a sequencing reaction with dATP, dCTP, dTTP, dGTP, and ddGTP?

* You would only be able to be determine where the cytosine were located in your original DNA template.

cDNA (Complementary DNA)

* called a cDNA because all the DNA in this library is complementary to mRNA). * Collection of clones containing all the DNA fragments from one source.

Which of the following best describes a cDNA library?

* consists of DNA sequences that are expressed

All of the following are differences between a dideoxyribonucleoside triphosphate (ddNTP) and a deoxyribonucleoside triphosphate (dNTP) that make ddNTPs useful in a sequencing reaction, EXCEPT _____.

* dNTPs have a base attached to their five-carbon ring

Where do restriction enzymes come from?

* from bacteria

What is one benefit of next-generation sequencing technologies over Sanger sequencing?

* next-generation sequencing technologies are faster

Taq polymerase

* polymerase DNA polymerase commonly used in PCR reactions. Isolated from the bacterium Thermus aquaticus, the enzyme is stable at high temperatures, so it is not denatured during the strand-separation step of the cycle.

Two basic approaches to amplifying a specific DNA fragment:

* replicating the DNA within cells (in vivo), * replicating the DNA enzymatically outside of cells (in vitro).

Briefly explain how synthetic probes are created to screen a DNA library when the protein encoded by the gene is known.

* synthetic probes can be created if the protein produced by the gene of interest has been isolated and its amino acid sequence has been determined. * Alternatively, synthetic probes can be created if the protein produced by the gene of interest has been isolated and its amino acid sequence has been determined.

DNA fragment must be amplified first , they req. 4 ingredients

1. Many copies of a primer that is complementary to one end of the target DNA strand 2. All four types of deoxyribonucleoside triphosphates, the normal precursors of DNA synthesis 3. A small amount of one of the four types of dideoxyribonucleoside triphosphates (ddATP, ddCTP, ddGTP, or ddTTP), which will terminate DNA synthesis as soon as it is incorporated into any growing chain (each of the four tubes receives a different ddNTP) 4. DNA polymerase