Restriction endonucleases, bacterial enzymes, are responsible for cleaving double-stranded DNA (dsDNA) into smaller fragments. These enzymes cleave at specific 4–8 base-pair sequences, known as restriction sites, thereby generating DNA segments called restriction fragments. The recognition sequences are palindromic in nature. Restriction enzymes can create either staggered cuts (sticky ends) or blunt-end cuts on the DNA molecule. Bacterial DNA ligases have the ability to join two DNA fragments from different sources if they have been cut by the same restriction endonuclease. This combination of two fragments is referred to as a recombinant DNA molecule. The introduction of a foreign DNA molecule into a replicating cell enables the amplification, or cloning, of the DNA, allowing for the production of numerous copies. To accomplish this, a vector, a DNA molecule to which the fragment of DNA to be cloned is joined, is necessary. Vectors must possess the ability to autonomously replicate within the host cell, contain at least one specific nucleotide sequence recognized by a restriction endonuclease, and carry at least one gene that confers the ability to select for the vector, such as an antibiotic resistance gene. Prokaryotic organisms typically contain small, circular, extrachromosomal DNA molecules called plasmids that can serve as vectors. These plasmids can be easily isolated from the bacterium or artificially constructed, joined with the DNA of interest, and reintroduced into the bacterium to allow for replication and the production of multiple copies of the hybrid plasmid. A DNA library refers to a collection of cloned restriction fragments from the DNA of an organism. In contrast, a genomic library consists of fragments of dsDNA obtained through the digestion of the organism's total DNA using a restriction endonuclease, followed by ligation to an appropriate vector. Ideally, a genomic library contains a copy of every DNA nucleotide sequence present in the genome. On the other hand, complementary DNA (cDNA) libraries only contain DNA sequences that are complementary to processed messenger RNA (mRNA) molecules found in a cell. These libraries vary based on cell type and environmental conditions. As cDNA lacks introns, it can be cloned into an expression vector to facilitate the synthesis of human proteins by bacteria or eukaryotes. Sequencing of cloned and purified DNA fragments can be achieved using methods like the Sanger dideoxy chain termination method. A probe, a small piece of RNA or single-stranded DNA, is used to identify a target DNA molecule of interest. Probes are usually labeled with a radioisotope, such as 32P, or another identifiable compound, such as biotin or a fluorescent dye. They have a nucleotide sequence that is complementary to the target DNA. Probes are valuable tools for identifying the clone of interest in a library or the specific band on a gel. Southern blotting is a technique employed to detect specific sequences in DNA. It involves cleaving the DNA with a restriction endonuclease, separating the resulting pieces through gel electrophoresis, denaturing them, and transferring (blotting) them onto a nitrocellulose membrane for analysis. A probe is used to detect the fragment of interest. The human genome contains numerous polymorphisms, which are DNA sequence variations at a given locus. Polymorphisms can arise from single-base changes or tandem repeats. Polymorphisms can serve as genetic markers that can be traced through families. A restriction fragment length polymorphism (RFLP) refers to a genetic variant that can be observed by cleaving the DNA into restriction fragments using a restriction enzyme. A base substitution at a restriction site can render the site unrecognizable by a particular restriction endonuclease. The same mechanism can also create a new restriction site. In either case, cleavage with the endonuclease produces fragments of varying lengths, which can be detected through hybridization with a probe. RFLP analysis is useful for diagnosing genetic diseases at an early stage of fetal development. The polymerase chain reaction (PCR), an alternative method for amplifying a specific DNA sequence, does not rely on biological cloning. PCR allows for the rapid synthesis of millions of copies of a particular nucleotide sequence within a few hours. It can amplify the sequence even when the target sequence constitutes less than one part in a million of the initial sample. PCR is applicable to amplifying DNA sequences from any source. The technique finds various applications, including 1) efficient comparison of a normal gene with a mutant form of the gene, 2) forensic analysis of DNA samples, 3) detection of low-abundance nucleic acid sequences, and 4) prenatal diagnosis and carrier detection of conditions like cystic fibrosis. mRNA and protein products of gene expression can be measured using techniques such as northern blots, which involve electrophoretic separation of a mixture of mRNA molecules in a sample, followed by hybridization with a radiolabeled probe. Microarrays are used to determine distinct patterns of gene expression in different cell types, such as normal and cancer cells. Enzyme-linked immunosorbent assays (ELISA) and western blots (immunoblots) are employed to detect specific proteins. Proteomics, the study of all proteins expressed by a genome, is a field of great interest. The goal of gene therapy is to insert a normal cloned gene to replace a defective gene in a somatic cell, while gene editing aims to repair a mutated gene. When a foreign gene (transgene) is inserted into the germline of an animal, a transgenic animal is created. Such animals can produce therapeutic proteins or serve as models for studying gene knockins or knockouts related to human diseases.
Restriction endonucleases are bacterial enzymes that cleave double-stranded DNA (dsDNA) into smaller fragments. Each enzyme cleaves at a specific 4–8 base-pair sequence (a restriction site), producing DNA segments called restriction fragments. The sequences that are recognized are palindromic. Restriction enzymes form either staggered cuts (sticky ends) or blunt-end cuts on the DNA. Bacterial DNA ligases can join two DNA fragments from different sources if they have been cut by the same restriction endonuclease. This hybrid combination of two fragments is called a recombinant DNA molecule. Introduction of a foreign DNA molecule into a replicating cell permits the amplification (production of many copies) of the DNA, a process called cloning. A vector is a molecule of DNA to which the fragment of DNA to be cloned is joined. Vectors must be capable of autonomous replication within the host cell, must contain at least one specific nucleotide sequence recognized by a restriction endonuclease, and must carry at least one gene that confers the ability to select for the vector such as an antibiotic resistance gene. Prokaryotic organisms normally contain small, circular, extrachromosomal DNA molecules called plasmids that can serve as vectors. They can be readily isolated from the bacterium (or artificially constructed), joined with the DNA of interest, and reintroduced into the bacterium, which will replicate, thus making multiple copies of the hybrid plasmid. A DNA library is a collection of cloned restriction fragments of the DNA of an organism. A genomic library is a collection of fragments of dsDNA obtained by digestion of the total DNA of the organism with a restriction endonuclease and subsequent ligation to an appropriate vector. It ideally contains a copy of every DNA nucleotide sequence in the genome. In contrast, complementary DNA (cDNA) libraries contain only those DNA sequences that are complementary to processed messenger RNA (mRNA) molecules present in a cell and differ according to cell type and environmental conditions. Because cDNA has no introns, it can be cloned into an expression vector for the synthesis of human proteins by bacteria or eukaryotes. Cloned, then purified, fragments of DNA can be sequenced, for example, using the Sanger dideoxy chain termination method. A probe is a small piece of RNA or single-stranded DNA (usually labeled with a radioisotope, such as 32P, or another identifiable compound, such as biotin or a fluorescent dye) that has a nucleotide sequence complementary to the DNA molecule of interest (target DNA). Probes can be used to identify which clone of a library or which band on a gel contains the target DNA. Southern blotting is a technique that can be used to detect specific sequences present in DNA. The DNA is cleaved using a restriction endonuclease, after which the pieces are separated by gel electrophoresis and are denatured and transferred (blotted) to a nitrocellulose membrane for analysis. The fragment of interest is detected using a probe. The human genome contains many thousands of polymorphisms (DNA sequence variations at a given locus). Polymorphisms can arise from single-base changes and from tandem repeats. A polymorphism can serve as a genetic marker that can be followed through families. A restriction fragment length polymorphism (RFLP) is a genetic variant that can be observed by cleaving the DNA into restriction fragments using a restriction enzyme. A base substitution in one or more nucleotides at a restriction site can render the site unrecognizable by a particular restriction endonuclease. A new restriction site also can be created by the same mechanism. In either case, cleavage with the endonuclease results in fragments of lengths differing from the normal that can be detected by hybridization with a probe. RFLP analysis can be used to diagnose genetic diseases early in the gestation of a fetus. The polymerase chain reaction (PCR), another method for amplifying a selected DNA sequence, does not rely on the biologic cloning method. PCR permits the synthesis of millions of copies of a specific nucleotide sequence in a few hours. It can amplify the sequence, even when the targeted sequence makes up less than one part in a million of the total initial sample. The method can be used to amplify DNA sequences from any source. Applications of the PCR technique include 1) efficient comparison of a normal gene with a mutant form of the gene, 2) forensic analysis of DNA samples, 3) detection of low-abundance nucleic acid sequences, and 4) prenatal diagnosis and carrier detection (for example, of cystic fibrosis). The products of gene expression (mRNA and proteins) can be measured by techniques such as northern blots, which are like Southern blots except that the sample contains a mixture of mRNA molecules that are separated by electrophoresis, then hybridized to a radiolabeled probe; microarrays are used to determine the differing patterns of gene expression in two different types of cells (for example, normal and cancer cells); enzyme-linked immunosorbent assays (ELISA); and western blots (immunoblots) are used to detect specific proteins. Proteomics is the study of all the proteins expressed by a genome. The goal of gene therapy is the insertion of a normal cloned gene to replace a defective gene in a somatic cell, whereas the goal of gene editing is the repair of a mutated gene. Insertion of a foreign gene (transgene) into the germline of an animal creates a transgenic animal that can produce therapeutic proteins or serve as gene knockin or knockout models for human diseases.