Genetics Lecture 21 Recombinant DNA Recombinant DNA In 1971, a paper published by Kathleen Danna and Daniel Nathans marked the beginning of the recombinant DNA era. The paper described the isolation of an enzyme from a bacterial strain and the use of the enzyme to cleave viral DNA at specific nucleotide sequences. It contained the first published photograph of DNA cut with such an enzyme, now called a restriction enzyme. 2 Using restriction enzymes and a number of other resources, researchers of the mid to late 1970s developed various techniques to create, replicate, and analyze recombinant DNA molecules DNA created by joining together pieces of DNA from different sources.. 3 1
The power of recombinant DNA technology is astonishing, enabling geneticists to identify and isolate a single gene or DNA segment of interest from a genome. Subsequently, through cloning, large quantities of identical copies of this specific DNA molecule can be produced. These identical copies, or clones, can then be manipulated for numerous purposes, including research into the structure and organization ofthe DNA, studying gene expression, and producing important commercial products from the protein encoded by a gene. The fundamental techniques involved in recombinant DNA technology subsequently led to the field of genomics, enabling scientists to sequence and analyze entire genomes. 4 Recombinant DNA Technology Although natural genetic processes such as crossing over produce recombined DNA molecules, the term recombinant DNA is generally reserved for molecules produced by artificially joining DNA obtained from different sources. The methods used to create these molecules were largely derived from nucleic acid biochemistry, coupled with genetic techniques developed for the study of bacteria and viruses. 5 Recombinant DNA technology needs two important tools used to construct and amplify recombinant DNA molecules: DNA cutting enzymes called restriction enzymes and DNA cloning vectors. The use of restriction enzymes and cloning vectors was largely responsible for advancing the field of molecular biology because of a wide range of techniques that are based on recombinant DNA technology. 6 2
Restriction Enzymes Cut DNA at Specific Recognition Sequences Restriction enzymes are produced by bacteria as a defense mechanism against infection by viruses. They restrict or prevent viral infection by degrading the DNA of invading viruses. More than 3500 restriction enzymes have been identified, and over 250 are commercially produced and available for use by researchers. A restriction enzyme recognizes and binds to DNA at a specific nucleotide sequence called a restriction site. The enzyme then cuts both strands of the DNA within that sequence by cleaving the phosphodiester backbone of DNA. Scientists commonly refer to this as digestion of DNA. 7 8 The usefulness of restriction enzymes in cloning derives from their ability to accurately and reproducibly cut genomic DNA into fragments. Restriction enzymes represent sophisticated molecular scissors for cutting DNA into fragments of desired sizes. The size of DNA restriction fragments produced by digesting DNA with a particular enzyme can be estimated from the number of times a given restriction enzyme cuts the DNA. Restriction sites are present randomly in the genome. Enzymes witha four base recognitionsequence sequence such such as the enzyme Alul, which recognizes the sequence AGCT will cut, on average, every 256 base pairs if all four nucleotides are present in equal proportions, producing many small fragments. 9 3
DNA Vectors Accept and Replicate DNA Molecules to Be Cloned Scientists recognized that DNA fragments produced by restriction enzyme digestion could be copied or cloned if they had a technique for replicating the fragments. The second key tool that allowed DNA cloning was the development of DNA cloning vectors. Vectors are DNA molecules that accept DNA fragments and replicate inserted DNA fragments when vectors are placed into host cells. 10 Many different vectors are available for cloning. Vectors differ in terms of the host cells they are able to enter and in the size of inserts they can carry, but most DNA vectors have several key properties. A vector contains several restriction sites that allow insertion of the DNA fragments to be cloned. Vectors must be introduced into host cells to allow for independent replication of the vector DNA and any DNA fragment it carries. To distinguish host cellsthat have taken up vectors from host cells that have not, the vector should carry a select able marker gene (usually an antibiotic resistance gene or the gene for an enzyme absent from the host cell). Many vectors incorporate specific sequences that allow for sequencing inserted DNA. 11 Ti Vectors For Plant Cells Introducing genes into plants is a common application that can be done in many ways. One widely used approach to insert genes into plant cells involves the soil bacterium Rhizobium radiobacter, which infects plant cells and produces tumors (called crown galls) in many species of plants. Rhizobium contains a plasmid called the Ti plasmid (tumor inducing), i and tumor formation is associated with ihthe presence of particular genes in the Ti plasmid. In the wild, when Ti plasmid carrying bacteria infect plant cells, a segment of the Ti plasmid, known as T DNA, is transferred into the genome of the host plant cell. Genes in the T DNA segment control tumor formation and the synthesis of compounds required for growth of the infecting bacteria. 12 4
Restriction sites in Ti plasmids can be used to insert foreign DNA, and recombinant vectors are introduced into Rhizobium by transformation. Tumor inducing genes from Ti plasmids are removed from the vector so that the recombinant vector does not result in tumor production. Rhizobium containing recombinant DNA is mixed with plant cells (not all types of plant cells can be infected by Rhizobium). Once inside the cell, the foreign DNA is inserted into the plant genome when the T DNA integrates into a host cell chromosome. Plant cells carrying a recombinant Ti plasmid can be grown in tissue culture. The presence of certain compounds in the culture medium plant cells stimulates the formation of roots and shoots, and eventually a mature plant carrying a foreign gene. 13 Host Cells for Cloning Vectors Besides deciding which DNA cloning vector to use, another cloning consideration is which host cells are used to accept recombinant DNA for cloning. There are many different reasons why particular host cells are chosen for a recombinant DNA experiment depending on the purpose of the work, E. coli is widely used as a prokaryotic host cell of choice when working with plasmids. The yeast Saccharomyces cerevisiae is extensively used as a host cell for the cloning and expression of eukaryotic genes. 14 The choice of host cells also extends to a number of other cell types, including insect cells. A variety of different human cell types can be grown in culture and used to express genes and proteins. Such cell lines can also then be subjected to various approaches for gene or protein ti functional analysis, including drug testing for effectiveness at blocking or influencing a particular recombinant protein being expressed, particularly if the cell lines are of a human disease condition such as cancer. 15 5
DNA Libraries Are Collections of Cloned Sequences Even when several hundred genes are introduced into larger vectors, one still needs a method for identifying thedna pieces thatwere cloned. DNA can be inserted into vectors and cloned a relatively straightforward process but we have not discussed how one knows what particular DNA sequence they have cloned. Simply cutting DNA and inserting into vectors does not tell you what gene or sequences have been cloned. 16 During the first several decades of DNA cloning, scientists created DNA libraries, which represent a collection of cloned DNA samples derived from a single source that could be a particular tissue type, cell type, or single individual. Depending on how a library is constructed, it may contain genes and noncoding regions of DNA. Generally there are two main types of libraries, genomic DNA libraries and complementary DNA (cdna) libraries. 17 Genomic Libraries Ideally, a genomic library consists of many overlapping fragments of the genome, with at least one copy of every DNA sequence in an organism s genome, which in summary span the entire genome. In making a genomic library DNA is extracted from cells or tissues and cut with restriction enzymes, and the resulting fragments are inserted into vectors. Since some vectors (such as plasmids) can carry only a few thousand base pairs of inserted DNA, selecting the vector so that the library contains the whole genome in the smallest number of clones is an important consideration. Because genomic DNA is the foreign DNA introduced into vectors, genomic libraries contain coding and noncoding segments of DNA. 18 6
Complementary DNA (cdna) Libraries cdna) libraries offer certain advantages over genomic libraries and continue to be a particularly useful methodology for gene cloning. This is primarily because a cdna library contains DNA copies called cdna which are made from the mrna molecules of a cell population and therefore represent the genes being expressed in the cells at the time the library was made. cdna is complementary to the nucleotide sequence of the mrna, and so unlike a genomic library, which contains all of the DNA in a genome gene coding and noncoding sequences a cdna library contains only expressed genes. These libraries compare expressed genes from normal tissues and diseased tissues. 19 The Polymerase Chain Reaction Is a Powerful Technique for copying DNA The recombinant DNA techniques developed in the early 1970s gave birth to the biotechnology industry because these methods enabled scientists to clone human genes, such as the insulin gene, whose protein product could be usedfor therapeutic purposes. However, cloning DNA using vectors and host cells is labor intensive and time consuming. In 1986, another technique, called the polymerase chain reaction (PCR), was developed. This advance revolutionized recombinant DNA methodology and further accelerated the pace of biological research. 20 PCR is a rapid method of DNA cloning that extends the power of recombinant DNA research and in many cases eliminates the need to use host cells for cloning. Although library cloning techniques still have specific applications of value, PCR is a method of choice for many applications, whether in molecular biology, human genetics, evolution, development, conservation, or forensics. 21 7
By copying a specific DNA sequence through a series of in vitro reactions, PCR can amplify target DNA sequences that are initially present in very small quantities in a population of other DNA molecules. PCR based DNA cloning has several advantages over library cloning approaches. PCR is rapid and can be carried out in a few hours, rather than the days required for making and screening DNA libraries. PCR is also very sensitive and amplifies specific DNA sequences from vanishingly small DNA samples, including the DNA in a single cell. A wide variety of PCR based techniques involve different variations of the basic technique described here. Several commonly used variations are used. 22 Limitations of PCR Although PCR is a valuable technique, it does have limitations: some information about the nucleotide sequence of the target DNA must be known in order to synthesize primers. In addition, even minor contamination of the sample with DNA from other sources can cause problems. For example, cells shed from the skin of a researcher can contaminate samples gathered from a crime scene or taken from fossils, making it difficult to obtain accurate results. 23 Applications of PCR Cloning DNA by PCR has been one of the most widely used techniques in genetics and molecular biology for over 20 years. PCR and its variations have many other applications as well. PCR is one of the most versatile techniques in modern genetics. Gene specific primers provide a way of using PCR for screening mutations involved in genetic disorders, allowing the location and nature of a mutation to be determined quickly. Primers can be designed to distinguish between target sequences that differ by only a single nucleotide. PCR is also a key diagnostic methodology for the detection of bacteria and viruses (such as hepatitis or HIV) in humans, and pathogenic bacteria such as E. coli and Staphylococcus aureus in contaminated food. 24 8
Molecular Techniques For Analyzing DNA The identification of genes and other DNA sequences by cloning or by PCR plays a very powerful role in analyzing genomic structure and function. In addition to cloning and PCR methods, a wide range of molecular techniques is available to geneticists, molecular biologists, and almost anyone who does research involving DNA and RNA, particularly those who study the structure, expression, and regulation of genes. 25 Restriction Mapping Historically, one of the first steps in characterizing a DNA clone was the construction of a restriction map. A restriction map establishes the number of order of and distances between restriction enzyme cleavage sites along a cloned segement of DNA, thus providing information about the length of the cloned insert and the location of restriction enzyme cleavage sites within the clone. 26 Nucleic Acid Blotting Several of the techniques rely on hybridization between complementary nucleic acid (DNA or RNA) molecules. One of the most widely used methods for detecting such hybrids is called Southern blotting. The Southern blot method can be used to identify which clones in a library contain a given DNA sequence and to characterize the size of the fragments. Southern blots can also be used to identify fragments carrying specific genes in genomic DNA digested with a restriction enzyme. Fragments of genomic clones isolated by Southern blots can in turn be isolated and recloned, pro viding a way to isolate parts of a gene. Southern blotting has also been a valuable tool for identifying the number of copies of a particular sequence or gene that are present in a genome. 27 9
DNA Sequencing The Ultimate Way to Characterize DNA Structure at the Molecular Level In a sense, a cloned DNA molecule or any DNA, from a single gene to an entire genome, is completely characterized at the molecular level only when its nucleotide sequence is known. The ability to sequence DNA has greatly enhanced our understanding of genome organization and increased our knowledge of gene structure, function, and mechanisms of regulation. Historically the most commonly used method of DNA sequencing was developed by Fred Sanger and his colleagues and is known as dideoxynucleotide chain termination sequencing or simply Sanger sequencing. 28 Sequencing Technologies Have Progressed Rapidly Nearly three decades since Fred Sanger was awarded part of the 1980 Nobel Prize in Chemistry (which he shared with Walter Gilbert and Paul Berg) for sequencing technology, DNA sequencing technologies have undergone an incredible evolution to dramatically improve sequencing capabilities. New innovations in sequencing technology are developing quickly. Sanger sequencing approaches (particularly those involving computer automated instruments such as capillary electrophoresis) still have their place in everyday routine applications that require sequencing, such as sequencing a relatively short piece of DNA amplified by PCR. 29 Next Generation Sequencing Technologies The development of genomics has spurred a demand for sequencers that are faster and capable of generating millions of bases of DNA sequences in a relatively short time, leading to the development of NGS approaches. Such sequencing technologies dispense with the Sanger technique and capillary electrophoresis methods in favor of sophisticated, parallel formats (simultaneous reaction formats) that use state of the art fluorescence imaging techniques. 30 10
??? of the day Name: 1. The power of recombinant DNA technology is astonishing, enabling geneticists to identify and isolate a single or DNA segment of interest from a genome. 2. Introducing genes into is a common application that can be done in many ways. 3. Depending on how a library is constructed, it may contain genes and regions of DNA. 4. Although is a valuable technique, it does have limitations: some information about the nucleotide sequence of the target DNA must be known in order to synthesize primers. 5. technologies are providing an unprecedented capacity for generating massive amounts of DNA sequence data rapidly and at dramatically reduced costs per base. 31 11