CHAPTER OUTLINE
Cloning Genomic DNA in YAC Vectors
Restriction Fragment Length Polymorphism Analysis
High-Yield Terms
Restriction endonuclease: any of a large family of enzymes that recognize, bind to, and hydrolyze specific nucleic acid sequences in double-stranded DNA
Cloning: in molecular biology this term refers to the production of large quantities of identical DNA molecules
Blotting: a molecular biological technique that involves the transfer of proteins, DNA, or RNA, out of the size-separating gel onto a solid support such as filter paper
DNA sequencing: the process of determining the precise order of nucleotides within a DNA molecule
Microarray: commonly known as DNA chip, is a collection of microscopic DNA spots attached to a solid surface
Gene chip: is the solid medium of a microarray containing the DNA spots
Transgenesis: the process of inserting a gene from one source into a living organism that would not normally contain it
Gene therapy: a technique involving the insertion of genes into an individual’s cells and tissues to treat a disease, such as a hereditary disease in which a deleterious mutant allele is replaced with a functional one
Introduction
Modern molecular medicine encompasses the utilization of many molecular biological techniques in the analysis of disease, disease genes, and disease gene function. The study of disease genes and their function in an unaffected individual has been possible by the development of recombinant DNA and cloning techniques. The basis of the term recombinant DNA refers to the recombining of different segments of DNA. Cloning refers to the process of preparing multiple copies of an individual type of recombinant DNA molecule. The classical mechanisms for producing recombinant molecules involves the insertion of exogenous fragments of DNA into either bacterially derived plasmid (circular double-stranded autonomously replicating DNAs found in bacteria) vectors or bacteriophage (viruses that infect bacteria)-based vectors. The term vector refers to the DNA molecule used to carry or transport DNA of interest into cells.
The technologies of molecular biology are critical to modern medical diagnosis and treatment. For example, cloning allows large quantities of therapeutically beneficial human proteins to be produced in pure form. In addition the cloning of genes allows for the utilization of the DNA for treatments referred to as gene therapy which involves the introduction of a normal copy of a gene into an individual harboring a defect gene. These molecular biology techniques involve the use of a wide array of different enzymes, many of which are purified from bacteria (Table 41-1).
Restriction Endonucleases
Restriction endonucleases are enzymes that will recognize, bind to, and hydrolyze specific nucleic acid sequences in double-stranded DNA (Table 41-2).
The key to the in vitro utilization of restriction endonucleases is their strict nucleotide sequence specificity. The different enzymes are identified by being given a name indicating the bacterium from which they were isolated; for example, the enzyme EcoRI, which recognizes the sequences, 5′–GAATTC–3′, was isolated from Escherichia coli. One unique feature of restriction enzymes is that the nucleotide sequences they recognize are palindromic, that is, they are the same sequences in the 5′ → 3′ direction of both strands. Some restriction endonucleases make staggered symmetrical cuts away from the center of their recognition site within the DNA duplex; some make symmetrical cuts in the middle of their recognition site while still others cleave the DNA at a distance from the recognition sequence. Enzymes that make staggered cuts leave the resultant DNA with cohesive or sticky ends (Figure 41-1). Enzymes that cleave the DNA at the center of the recognition sequence leave blunt-ended fragments of DNA.
FIGURE 41-1: Results of restriction endonuclease digestion. Digestion with a restriction endonuclease can result in the formation of DNA fragments with sticky, or cohesive, ends (A) or blunt ends (B); phosphodiester backbone, black lines; interstrand hydrogen bonds between purine and pyrimidine bases, blue. This is an important consideration in devising cloning strategies. Murray RK, Bender DA, Botham KM, Kennelly PJ, Rodwell VW, Weil PA. Harper’s Illustrated Biochemistry. 29th ed. New York: McGraw-Hill; 2012.
Any 2 pieces of DNA containing the same sequences within their sticky ends can anneal together and be covalently ligated together in the presence of DNA ligase. Any 2 blunt-ended fragments of DNA can be ligated together irrespective of the sequences at the ends of the duplexes.
Cloning DNA
Any fragment of DNA can be cloned once it is introduced into a suitable vector for transforming an applicable host cell. Cloning refers to the production of large quantities of identical DNA molecules and usually involves the use of a bacterial cell as a host for the DNA, although cloning can be done in eukaryotic cells as well. cDNA cloning refers to the production of a library of cloned DNAs that represent all mRNAs present in a particular cell or tissue. Genomic cloning refers to the production of a library of cloned DNAs representing the entire genome of a particular organism. From either of these types of libraries one can isolate (by a variety of screening protocols) a single cDNA or a gene clone.
In order to clone either cDNAs or copies of genes a vector is required to carry the cloned DNA. Vectors used in molecular biology are of 2 basic classes. One class of vectors is derived from bacterial plasmids (Figure 41-2). Plasmids are circular DNAs found in bacteria that replicate autonomously from the host genome. These DNAs were first identified because they harbored genes that conferred antibiotic resistance to the bacteria. The antibiotic resistance genes found on the original plasmids are used in modern in vitro engineered plasmids to allow selection of bacteria that have taken up the plasmids containing the DNAs of interest. Plasmids are limited in that in general fragments of DNA less than 10,000 base pairs (bp) can be cloned (Table 41-3).
FIGURE 41-2: Typical plasmid vector cloning. A plasmid cloning vector is digested with the same enzymes (EcoRI and PvuII) as the target DNA. Most cloning plasmids have a section of unique restriction endonuclease sites called the multicloning site (MCS). The target DNA is ligated into the plasmid with DNA ligase with the result being a recombinant plasmid that can be used to transfect bacteria. Reproduced with permission of themedicalbiochemistrypage, LLC.
The second class of vector is derived from the bacteriophage (bacterial virus) lambda. The advantage to lambda-based vectors is that they can carry fragments of DNA up to 25,000 bp. In the analysis of the human genome even lambda-based vectors are limiting and a yeast artificial chromosome (YAC) vector system has been developed for the cloning of DNA fragments from 500,000 bp to 3,000,000 bp.
The term restriction endonuclease was given to this class of bacterially derived enzymes since they were identified as being involved in restricting the growth of certain bacteriophages via a process referred to as modification and restriction.
cDNA Cloning
cDNAs are made from the mRNAs of a cell by any number of related techniques (Figure 41-3). Each technique consists of first reverse transcription of the mRNA followed by synthesis of the second strand of DNA and insertion of the double-stranded cDNA into either a plasmid or lambda vector for cloning. This process creates a library of cloned cDNA representing each mRNA species. Screening of the library for a particular cDNA clone is accomplished using nucleic acid or protein-based (proteins or antibodies) probes. cDNA libraries can also be screened by biological assay of the products produced by the cloned cDNAs. Screening with proteins, antibodies, or by biological assay are mechanisms for analysis of the expression of proteins from cloned cDNAs and is given the term expression cloning. Nucleic acids probes can be generated from DNA (including synthetic oligonucleotides, oligos) or RNA. Nucleic acid probes can be radioactively labeled or labeled with modified nucleotides that are recognizable by specific antibodies and detected by colorimetric or chemiluminescent assays.
FIGURE 41-3: Typical process for production and cloning of cDNA. This example shows the use of a specific primer-adapter containing the sequences for the restriction enzyme NotI in addition to the poly(T) for annealing to the poly(A) tail of the RNA. It is possible to use only poly(T) or poly(T) with other restriction sites or random primers (a mixture of oligos that contain random sequences) to initiate the first strand cDNA reaction. In some cases poly(T) priming does not allow for extension of the cDNA to the 5′-end of the RNA, the use of random primers can overcome this problem since they will prime first strand synthesis all along the mRNA. This technique shows the ligation of EcoRI adapters followed by EcoRI and NotI digestion. This process allows the cDNAs to all be cloned in one direction, termed directional cloning. Reproduced with permission of themedicalbiochemistrypage, LLC.
Genomic Cloning
The majority of genomic cloning utilizes lambda-based vector systems. These vector systems are capable of carrying 15,000 to 25,000 bp of DNA. Still larger genomic DNA fragments can be cloned into YAC vectors (see below). Genomic DNA can be isolated and cloned from any nucleated cell. The genomic DNA is first digested with restriction enzymes to generate fragments in the size range that are optimal for the vector being utilized for cloning. Given that some genes encompass many more base pairs than can be inserted into a given vector, the clones that are present in a genomic library must be overlapping (Figure 41-4). In order to generate overlapping clones, the DNA is only partially digested with restriction enzymes. This means that not every restriction site, present in all the copies of a given gene in the preparation of DNA, is cleaved. The partially digested DNA is then size-selected by a variety of techniques (eg, gel electrophoresis or gradient centrifugation) prior to cloning. Screening of genomic libraries is accomplished primarily with nucleic acid-based probes. However, they can be screened with proteins that are known to bind specific sequences of DNA (eg, transcription factors).
FIGURE 41-4: Diagrammatic representation of cloning genomic DNA. The boxes indicate exons and the lines separating the boxes represent introns. The bold arrows indicate the positions of restriction enzyme sites, for example, Sau3AI. Following partial enzyme digestion a wide range of different fragments of the gene will be generated; 4 possible fragments are indicated. Fragments in the size range of 15 to 25 kilobase pairs (kbp) are purified by gel electrophoresis or gradient centrifugation and ligated into a lambda vector. The DNA is packaged into phage particles in vitro and used to infect E coli. Reproduced with permission of themedicalbiochemistrypage, LLC.
Cloning Genomic DNA in YAC Vectors
YAC vectors allow the cloning, within yeast cells, fragments of genomic DNA that approach 3,000,000 bp (Figure 41-5). These vectors contain several elements of typical yeast chromosomes, hence the term YAC. The YAC vectors contain a yeast centromere (CEN), yeast telomeres (TEL), and a yeast autonomously replicating sequence (ARS). Yeast ARS are essentially origins of replication that function in yeast cells autonomously from the replication of yeast chromosomal replication origins. YAC vectors also contain genes (eg, URA3, a gene involved in uracil synthesis) that allow selection of yeast cells that have taken up the vector. In order to propagate the vector in bacterial cells, prior to insertion of genomic DNA, YAC vectors contain a bacterial replication origin and a bacterial selectable marker such as the gene for ampicillin resistance.
FIGURE 41-5: Diagrammatic representation of a typical YAC vector used to clone genomic DNA. The vector contains yeast telomeres (TEL), a centromere (CEN), a selectable marker (URA3), and autonomously replicating sequences (ARS) as well as bacterial plasmid sequences for antibiotic selection and replication in E coli. PFGE, pulsed field gel electrophoresis. Reproduced with permission of themedicalbiochemistrypage, LLC.
Analysis of Cloned Products
The analysis of cloned cDNAs and genes involves a number of techniques. The initial characterization usually involves mapping of the number and location of different restriction enzyme sites. This information is useful for DNA sequencing since it provides a means to digest the clone into specific fragments for subcloning, a process involving the cloning of fragments of a particular cloned DNA. Once the DNA is fully characterized cDNA clones can be used to produce RNA in vitro and the RNA translated in vitro to characterize the encoded protein. Clones of cDNAs also can be used as probes to analyze the structure of a gene by Southern blotting or to analyze the size of the RNA and pattern of its expression by Northern blotting (Figure 41-6). Northern blotting is also a useful tool in the analysis of the exon-intron organization of gene clones since only fragments of a gene that contain exons will hybridize to the RNA on the blot. Proteins in tissues or those expressed by cloned cDNA can be analyzed by the technique of Western blotting.
FIGURE 41-6: The blot transfer procedure. In a Southern, or DNA blot transfer, DNA isolated from a cell line or tissue is digested with one or more restriction enzymes. This mixture is pipetted into a well in an agarose or polyacrylamide gel and exposed to a direct electrical current. DNA, being negatively charged, migrates toward the anode; the smaller fragments move the most rapidly. After a suitable time, the DNA within the gel is denatured by exposure to mild alkali and transferred to nitrocellulose or nylon paper, resulting in an exact replica of the pattern on the gel, by the blotting technique devised by Southern. The DNA is bound to the paper by exposure to heat or UV, and the paper is then exposed to the labeled cDNA probe, which hybridizes to complementary strands on the filter. After thorough washing, the paper is exposed to x-ray film or an imaging screen, which is developed to reveal several specific bands corresponding to the DNA fragment that recognized the sequences in the cDNA probe. The RNA, or Northern, blot is conceptually similar. RNA is subjected to electrophoresis before blot transfer. This requires some different steps from those of DNA transfer, primarily to ensure that the RNA remains intact, and is generally somewhat more difficult. In the protein, or Western, blot, proteins are electrophoresed and transferred to special paper that avidly binds proteins and then probed with a specific antibody or other probe molecule. (Asterisks signify labeling, either radioactive or fl uorescent.) In the case of Southwestern blotting (see the text; not shown), a protein blot similar to that shown above under “Western” is exposed to labeled nucleic acid, and protein-nucleic acid complexes formed are detected by autoradiography or imaging. Murray RK, Bender DA, Botham KM, Kennelly PJ, Rodwell VW, Weil PA. Harper’s Illustrated Biochemistry. 29th ed. New York: McGraw-Hill; 2012.
Southern Blotting
The DNA to be analyzed is first digested with a given restriction enzyme, then the resultant DNA fragments are separated in an agarose gel. The DNA is transferred from the gel to a solid support, such as a nylon filter, by either capillary diffusion or under electric current. The DNA is fixed to the filter by baking or ultraviolet light treatment. The filter can then be probed for the presence of a given fragment of DNA by various radioactive or nonradioactive means.
Northern Blotting
Northern blotting involves the analysis of RNA following its attachment to a solid support. The RNA is sized by gel electrophoresis then transferred to nitrocellulose or nylon filter paper as for Southern blotting. Probing the filter for a particular RNA is done similarly to probing of Southern blots.
Western Blotting
Western blotting involves the analysis of proteins following attachment to a solid support. The proteins are separated by size SDS-PAGE and electrophoretically transferred to nitrocellulose or nylon filters. The filter is then probed with antibodies raised against a particular protein or they can be probed with DNA probes if analyzing DNA-binding proteins.
DNA Sequencing
Sequencing of DNA can be accomplished by either chemical or enzymatic means. The original technique for sequencing, Maxam-Gilbert sequencing, relies on the nucleotide-specific chemical cleavage of DNA and is not routinely used any more. The enzymatic technique, Sanger sequencing, involves the use of dideoxynucleotides (2′,3′-dideoxy) that terminate DNA synthesis and is, therefore, also called dideoxy chain termination sequencing (Figure 41-7).
FIGURE 41-7: Sequencing of DNA by the chain termination method devised by Sanger. The ladder-like arrays represent from bottom to top all of the successively longer fragments of the original DNA strand. Knowing which specific dideoxynucleotide reaction was conducted to produce each mixture of fragments, one can determine the sequence of nucleotides from the unlabeled end toward the labeled end (*) by reading up the gel. The base-pairing rules of Watson and Crick (A-T, G-C) dictate the sequence of the other (complementary) strand. (Asterisks signify the site of radiolabeling.) Shown (left, middle) are the terminated synthesis products of a hypothetical fragment of DNA, sequence shown. An autoradiogram (right) of an actual set of DNA sequencing reactions that utilized the four 32P-labeled dideoxynucleotides indicated at the top of the scanned autoradiogram (ie, dideoxy(dd)G, ddA, ddT, ddC). Electrophoresis was from top to bottom. The deduced DNA sequence is listed on the right side of the gel. Note the log-linear relationship between distance of migration (ie, top to bottom of gel) and DNA fragment length. Current state-of-the-art DNA sequencers no longer utilize gel electrophoresis for fractionation of labeled synthesis products. Moreover in the NGS sequencing platforms, synthesis is followed by monitoring incorporation of the four fluorescently labeled dNTPs. Murray RK, Bender DA, Botham KM, Kennelly PJ, Rodwell VW, Weil PA. Harper’s Illustrated Biochemistry. 29th ed. New York: McGraw-Hill; 2012.
Southern blotting is the analysis of DNA structure following its attachment to a solid support. This technique is called Southern blotting as it was developed by Edwin Southern.
High-Yield Concept
The polymerase chain reaction (PCR) is a powerful technique used to amplify DNA millions of fold, by repeated replication of a template, in a short period of time (Figure 41-8). The process utilizes sets of specific in vitro synthesized oligonucleotides to prime DNA synthesis.
FIGURE 41-8: The polymerase chain reaction is used to amplify specific gene sequences. Double-stranded DNA is heated to separate it into individual strands. These bind two distinct primers that are directed at specific sequences on opposite strands and that define the segment to be amplified. DNA polymerase extends the primers in each direction and synthesizes 2 strands complementary to the original 2. This cycle is repeated several times, giving an amplified product of defined length and sequence. Note that the 2 primers are present in vast excess. Murray RK, Bender DA, Botham KM, Kennelly PJ, Rodwell VW, Weil PA. Harper’s Illustrated Biochemistry. 29th ed. New York: McGraw-Hill; 2012.
Diagnostic Methodologies
The Polymerase Chain Reaction
The polymerase chain reaction (PCR) constitutes a high yield in vitro DNA synthesis technique that requires synthetic oligonucleotide primers. The design of the primers is dependent upon the sequences of the DNA that is to be amplified. The technique is carried out through many cycles (usually 20-50) of melting the DNA template at high temperature, allowing the primers to anneal to complimentary sequences within the template and then replicating the template with DNA polymerase. The process has been automated with the use of thermostable DNA polymerases. During the first round of replication a single copy of DNA is converted to 2 copies and so on resulting in an exponential increase in the number of copies of the sequences targeted by the primers. After just 20 cycles a single copy of DNA is amplified over 2,000,000 fold. The products of PCR reactions are generally analyzed by separation in agarose gels followed by ethidium bromide staining and visualization with UV transillumination.
PCR can be used in the analysis of disease genes by being able to amplify detectable amounts of specific fragments of DNA. The amplified fragments from disease genes may be larger, due to insertions, or smaller, due to deletions. The dramatic amplification of DNA by PCR allows the analysis of disease genes in extremely small samples of DNA. For example, only a single fetal cell need be extracted from amniotic fluid in order to analyze for the presence of specific disease genes. The PCR technique also can be used to identify the level of expression of genes in extremely small samples of material, for example, tissues or cells from the body. This technique is termed reverse transcription-PCR (RT-PCR) because the mRNA is first converted into single-stranded DNA by reverse transcriptase and then the DNA is amplified.
The RT-PCR technique is most frequently utilized in a process referred to as quantitative PCR (qPCR) or real-time PCR. In the qPCR technique the amplification is done in the presence of a fluorophore whose spectrum of light emission can be detected by the qPCR machine. The easiest and least expensive method involves a fluorescent dye that intercalates into the DNA and thus the level of fluorescence is directly proportional to the amount of DNA present at any given time. The more sensitive method involves the use of an oligo with a fluorophore attached but is unable to emit light in this form. The oligo is designed to hybridize to sequences between the 2 amplification primers. During DNA synthesize the fluorophore-tagged oligo is degraded and when released the fluorophore emits light at a specific wavelength. The increase in fluorescence is detected while the PCR reactions are occurring, hence the term real-time PCR. In addition, the level of fluorescence can be compared to an amplifiable standard DNA template allowing for quantitative comparison of the amount of amplification of a particular gene of interest.
Restriction Fragment Length Polymorphism Analysis
The genetic variability at a particular locus due to even single base changes can alter the pattern of restriction enzyme digestion fragments that can be generated. Restriction fragment length polymorphism (RFLP) analysis takes advantage of this and utilizes Southern blotting of restriction enzyme digested genomic DNA or more commonly, PCR products, to detect familial patterns of the fragments of a given gene. A classic example of a disease detectable by RFLP is sickle cell anemia (Figure 41-9).
FIGURE 41-9: Pedigree analysis of sickle cell disease. The top part of the figure (A) shows the first part of the β-globin gene and the MstII restriction enzyme sites in the normal (A) and sickle-cell (S) β-globin genes. Digestion with the restriction enzyme MstII results in DNA fragments 1.15 kb and 0.2 kb long in normal individuals. The T-to-A change in individuals with sickle cell disease abolishes 1 of the 3 MstII sites around the β-globin gene; hence, a single restriction fragment 1.35 kb in length is generated in response to MstII. This size difference is easily detected on a Southern blot. (The 0.2-kb fragment would run off the gel in this illustration.) (B) Pedigree analysis shows 3 possibilities: AA = normal (open circle); AS = heterozygous (half-solid circles, half-solid square); SS = homozygous (solid square). This approach can allow for prenatal diagnosis of sickle-cell disease (dash-sided square). Murray RK, Bender DA, Botham KM, Kennelly PJ, Rodwell VW, Weil PA. Harper’s Illustrated Biochemistry. 29th ed. New York: McGraw-Hill; 2012.
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