Tools of Genetic Engineering

Recombinant DNA Technology

Molecular cloning permits the replication of a specific DNA sequence in a living microorganism.

Learning Objectives

Show some of the methods and uses of recombinant DNA

Key Takeaways

Key Points

  • Although a very large number of host organisms and molecular cloning vectors are in use, the great majority of molecular cloning experiments begin with a laboratory strain of the bacterium E. coli (Escherichia coli) and a plasmid cloning vector.
  • E. coli and plasmid vectors are in common use because they are technically sophisticated, versatile, widely available, and offer rapid growth of recombinant organisms with minimal equipment.
  • Modern bacterial cloning vectors (e.g. pUC19) use the blue-white screening system to distinguish colonies ( clones ) of transgenic cells from those that contain the parental vector.

Key Terms

  • polymerase chain reaction: A technique in molecular biology for creating multiple copies of DNA from a sample; used in genetic fingerprinting etc.
  • molecular cloning: a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms.
  • restriction enzyme: An endonuclease that catalyzes double-strand cleavage of DNA containing a specific sequence.

Recombinant DNA technology also referred to as molecular cloning is similar to polymerase chain reaction ( PCR ) in that it permits the replication of a specific DNA sequence. The fundamental difference between the two methods is that molecular cloning involves replication of the DNA in a living microorganism, while PCR replicates DNA in an in vitro solution, free of living cells.

In standard molecular cloning experiments, the cloning of any DNA fragment essentially involves seven steps:

  1. Choice of host organism and cloning vector
  2. Preparation of vector DNA
  3. Preparation of DNA to be cloned
  4. Creation of recombinant DNA
  5. Introduction of recombinant DNA into host organism
  6. Selection of organisms containing recombinant DNA
  7. Screening for clones with desired DNA inserts and biological properties

Although a very large number of host organisms and molecular cloning vectors are in use, the great majority of molecular cloning experiments begin with a laboratory strain of the bacterium E. coli (Escherichia coli) and a plasmid cloning vector. E. coli and plasmid vectors are in common use because they are technically sophisticated, versatile, widely available, and offer rapid growth of recombinant organisms with minimal equipment. The cloning vector is treated with a restriction endonuclease to cleave the DNA at the site where foreign DNA will be inserted. The restriction enzyme is chosen to generate a configuration at the cleavage site that is compatible with that at the ends of the foreign DNA.

Typically, this is done by cleaving the vector DNA and foreign DNA with the same restriction enzyme, for example EcoRI. Most modern vectors contain a variety of convenient cleavage sites that are unique within the vector molecule (so that the vector can only be cleaved at a single site) and is located within a gene (frequently beta-galactosidase) whose inactivation can be used to distinguish recombinant from non-recombinant organisms at a later step in the process. To improve the ratio of recombinant to non-recombinant organisms, the cleaved vector may be treated with an enzyme (alkaline phosphatase) that dephosphorylates the vector ends. Vector molecules with dephosphorylated ends are unable to replicate, and replication can only be restored if foreign DNA is integrated into the cleavage site.

For cloning of genomic DNA, the DNA to be cloned is extracted from the organism of interest. Polymerase chain reaction (PCR) methods are often used for amplification of specific DNA or RNA (RT-PCR) sequences prior to molecular cloning. The purified DNA is then treated with a restriction enzyme to generate fragments with ends capable of being linked to those of the vector. If necessary, short double-stranded segments of DNA (linkers) containing desired restriction sites may be added to create end structures that are compatible with the vector. The creation of recombinant DNA is in many ways the simplest step of the molecular cloning process. DNA prepared from the vector and foreign source are simply mixed together at appropriate concentrations and exposed to an enzyme (DNA ligase) that covalently links the ends together. This joining reaction is often termed ligation. The resulting DNA mixture containing randomly joined ends is then ready for introduction into the host organism. The DNA mixture, previously manipulated in vitro, is moved back into a living cell, referred to as the host organism. The methods used to get DNA into cells are varied, and the name applied to this step in the molecular cloning process will often depend upon the experimental method that is chosen (e.g. transformation, transduction, transfection, electroporation).

When microorganisms are able to take up and replicate DNA from their local environment, the process is termed transformation, and cells that are in a physiological state such that they can take up DNA are said to be competent. When bacterial cells are used as host organisms, the selectable marker is usually a gene that confers resistance to an antibiotic that would otherwise kill the cells, typically ampicillin. Cells harboring the vector will survive when exposed to the antibiotic, while those that have failed to take up vector sequences will die. Modern bacterial cloning vectors (e.g. pUC19) use the blue-white screening system to distinguish colonies (clones) of transgenic cells from those that contain the parental vector.

In these vectors, foreign DNA is inserted into a sequence that encodes an essential part of beta-galactosidase, an enzyme whose activity results in formation of a blue-colored colony on the culture medium that is used for this work. Insertion of the foreign DNA into the beta-galactosidase coding sequence disables the function of the enzyme, so that colonies containing recombinant plasmids remain colorless (white). Therefore, recombinant clones are easily identified.

image

Blue White screen: The blue-white screen is a screening technique that allows for the detection of successful ligations in vector-based gene cloning. DNA of interest is ligated into a vector. The vector is then transformed into competent cell (bacteria). The competent cells are grown in the presence of X-gal. If the ligation was successful, the bacterial colony will be white; if not, the colony will be blue. This technique allows for the quick and easy detection of successful ligation.

Selection

A selectable marker is usually a gene that confers resistance to an antibiotic that would otherwise kill the cells.

Learning Objectives

Identify the purpose of selection in genetic engineering

Key Takeaways

Key Points

  • Recombinant DNA is introduced into the organism from which the replication sequences were obtained, then the foreign DNA will be replicated along with the host cell’s DNA in the transgenic organism.
  • Artificial genetic selection is the process in which cells that have not taken up DNA are selectively killed, and only those cells that can actively replicate DNA containing the selectable marker gene encoded by the vector are able to survive.
  • When bacterial cells are used as host organisms, the selectable marker is usually a gene that confers resistance to an antibiotic that would otherwise kill the cells, typically ampicillin.

Key Terms

  • molecular cloning: a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms.
  • PCR: polymerase chain reaction

Scientists who do experimental genetics employ artificial selection experiments that permit the survival of organisms with user-defined phenotypes. Artificial selection is widely used in the field of microbial genetics, especially molecular cloning.

DNA recombination has been used to create gene replacements, deletions, insertions, inversions. Gene cloning and gene/protein tagging is also common. For gene replacements or deletions, usually a cassette encoding a drug-resistance gene is made by PCR.

Molecular cloning is a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms. The use of the word cloning refers to the fact that the method involves the replication of a single DNA molecule starting from a single living cell to generate a large population of cells containing identical DNA molecules. Molecular cloning generally uses DNA sequences from two different organisms: the species that is the source of the DNA to be cloned, and the species that will serve as the living host for replication of the recombinant DNA. Molecular cloning methods are central to many contemporary areas of modern biology and medicine.

In a conventional molecular cloning experiment, the DNA to be cloned is obtained from an organism of interest. It is then treated with enzymes in the test tube to generate smaller DNA fragments. Subsequently, these fragments are then combined with vector DNA to generate recombinant DNA molecules. The recombinant DNA is then introduced into a host organism (typically an easy-to-grow, benign, laboratory strain of E. coli bacteria ). This will generate a population of organisms in which recombinant DNA molecules are replicated along with the host DNA. Because they contain foreign DNA fragments, these are transgenic or genetically-modified microorganisms (GMO). This process takes advantage of the fact that a single bacterial cell can be induced to take up and replicate a single recombinant DNA molecule. This single cell can then be expanded exponentially to generate a large amount of bacteria, each of which contain copies of the original recombinant molecule. Thus, both the resulting bacterial population, and the recombinant DNA molecule, are commonly referred to as “clones”. Strictly speaking, recombinant DNA refers to DNA molecules, while molecular cloning refers to the experimental methods used to assemble them.

Molecular cloning takes advantage of the fact that the chemical structure of DNA is fundamentally the same in all living organisms. Therefore, if any segment of DNA from any organism is inserted into a DNA segment containing the molecular sequences required for DNA replication, and the resulting recombinant DNA is introduced into the organism from which the replication sequences were obtained, then the foreign DNA will be replicated along with the host cell’s DNA in the transgenic organism.

Molecular cloning is similar to polymerase chain reaction (PCR) in that it permits the replication of a specific DNA sequence. The fundamental difference between the two methods is that molecular cloning involves replication of the DNA in a living microorganism, while PCR replicates DNA in an in vitro solution, free of living cells. Whichever method is used, the introduction of recombinant DNA into the chosen host organism is usually a low efficiency process; that is, only a small fraction of the cells will actually take up DNA. Experimental scientists deal with this issue through a step of artificial genetic selection, in which cells that have not taken up DNA are selectively killed, and only those cells that can actively replicate DNA containing the selectable marker gene encoded by the vector are able to survive. When bacterial cells are used as host organisms, the selectable marker is usually a gene that confers resistance to an antibiotic that would otherwise kill the cells, typically ampicillin. Cells harboring the vector will survive when exposed to the antibiotic, while those that fail to take up vector sequences die. When mammalian cells (e.g. human or mouse cells) are used, a similar strategy is used, except that the marker gene (in this case typically encoded as part of the kanMX cassette) confers resistance to the antibiotic Geneticin.

image

Plasmid vector map: This vector confers amplicillin resistance.

Mutation

Mutations are accidental changes in a genomic sequence of DNA; this includes the DNA sequence of a cell’s genome or the DNA or RNA sequence.

Learning Objectives

Explain genetic manipulation through mutations

Key Takeaways

Key Points

  • Mutations are caused by radiation, viruses, transposons, and mutagenic chemicals. They are also caused by errors that occur during meiosis or DNA replication.
  • Site-directed mutagenesis, also called site-specific mutagenesis or oligonucleotide-directed mutagenesis, is a molecular biology technique often used in biomolecular engineering in which a mutation is created at a defined site in a DNA molecule.
  • The basic procedure requires the synthesis of a short DNA primer. This synthetic primer contains the desired mutation. It is complementary to the template DNA around the mutation site so it can hybridize with the DNA in the gene of interest.

Key Terms

  • site-directed mutagenesis: Also called site-specific mutagenesis or oligonucleotide-directed mutagenesis, is a molecular biology technique often used in biomolecular engineering in which a mutation is created at a defined site in a DNA molecule.
  • mutation: Any heritable change of the base-pair sequence of genetic material.

In molecular biology and genetics, mutations are accidental changes in a genomic sequence of DNA: the DNA sequence of a cell’s genome or the DNA or RNA sequence in some viruses. These random sequences can be defined as sudden and spontaneous changes in the cell. Mutations are caused by radiation, viruses, transposons, and mutagenic chemicals. They are also caused by errors that occur during meiosis or DNA replication. They can also be induced by the organism itself, through cellular processes such as hypermutation.

Site-directed mutagenesis, also called site-specific mutagenesis or oligonucleotide-directed mutagenesis, is a molecular biology technique often used in biomolecular engineering in which a mutation is created at a defined site in a DNA molecule. In general, this form of mutagenesis requires that the wild type gene sequence be known. It is commonly used in protein engineering.

image

The distribution of fitness effects: The distribution of fitness effects of mutations in vesicular stomatitis virus. In this experiment, random mutations were introduced into the virus by site-directed mutagenesis. The fitness of each mutant was compared with the ancestral type. A fitness of zero, less than one, one, more than one, respectively, indicates that mutations are lethal, deleterious, neutral, and advantageous.

The basic procedure requires the synthesis of a short DNA primer. This synthetic primer contains the desired mutation and is complementary to the template DNA around the mutation site so it can hybridize with the DNA in the gene of interest. The mutation may be a single base change (a point mutation), multiple base changes, deletion, or insertion. The single-stranded primer is then extended using a DNA polymerase, which copies the rest of the gene. The copied gene contains the mutated site. It is then introduced into a host cell as a vector and cloned. Finally, mutants are selected.

The original method using single-primer extension was inefficient due to a lower yield of mutants. The resulting mixture may contain both the original unmutated template as well as the mutant strand, producing a mix population of mutant and non-mutant progenies. The mutants may also be counter-selected due to presence of a mismatch repair system which favors the methylated template DNA. Many approaches have since been developed to improve the efficiency of mutagenesis.

Reproductive Cloning

Reproductive cloning, possible through artificially-induced asexual reproduction, is a method used to make a clone of an entire organism.

Learning Objectives

Differentiate reproductive cloning from cellular and molecular cloning

Key Takeaways

Key Points

  • A form of asexual reproduction, parthenogenesis, occurs when an embryo grows and develops without the fertilization of the egg.
  • In reproductive cloning, if the haploid nucleus of an egg cell is replaced with a diploid nucleus from the cell of an individual of the same species, it will become a zygote that is genetically identical to the donor.
  • Reproductive cloning has become successful, but still has limitations as cloned individuals often exhibit facial, limb, and cardiac abnormalities.
  • Therapeutic cloning, the cloning of human embryos as a source of embryonic stem cells, has been attempted in order to produce cells that can be used to treat detrimental diseases or defects.

Key Terms

  • clone: a living organism produced asexually from a single ancestor, to which it is genetically identical
  • stem cell: a primal undifferentiated cell from which a variety of other cells can develop through the process of cellular differentiation
  • parthenogenesis: a form of asexual reproduction where growth and development of embryos occur without fertilization

Reproductive Cloning

Reproductive cloning is a method used to make a clone or an identical copy of an entire multicellular organism. Most multicellular organisms undergo reproduction by sexual means, which involves genetic hybridization of two individuals (parents), making it impossible to generate an identical copy or clone of either parent. Recent advances in biotechnology have made it possible to artificially induce asexual reproduction of mammals in the laboratory.

image

Reproductive Cloning of Dolly, the Sheep: Dolly the sheep was the first mammal to be cloned. To create Dolly, the nucleus was removed from a donor egg cell. The nucleus from a second sheep was then introduced into the cell, which was allowed to divide to the blastocyst stage before being implanted in a surrogate mother.

Parthenogenesis, or “virgin birth,” occurs when an embryo grows and develops without the fertilization of the egg occurring; this is a form of asexual reproduction. An example of parthenogenesis occurs in species in which the female lays an egg. If the egg is fertilized, it is a diploid egg and the individual develops into a female; if the egg is not fertilized, it remains a haploid egg and develops into a male. The unfertilized egg is called a parthenogenic, or virgin, egg. Some insects and reptiles lay parthenogenic eggs that can develop into adults.

Sexual reproduction requires two cells; when the haploid egg and sperm cells fuse, a diploid zygote results. The zygote nucleus contains the genetic information to produce a new individual. However, early embryonic development requires the cytoplasmic material contained in the egg cell. This idea forms the basis for reproductive cloning. If the haploid nucleus of an egg cell is replaced with a diploid nucleus from the cell of any individual of the same species (called a donor), it will become a zygote that is genetically identical to the donor. Somatic cell nuclear transfer is the technique of transferring a diploid nucleus into an enucleated egg. It can be used for either therapeutic cloning or reproductive cloning.

The first cloned animal was Dolly, a sheep who was born in 1996. The success rate of reproductive cloning at the time was very low. Dolly lived for seven years and died of respiratory complications. There is speculation that because the cell DNA belongs to an older individual, the age of the DNA may affect the life expectancy of a cloned individual. Since Dolly, several animals (e.g. horses, bulls, and goats) have been successfully cloned, although these individuals often exhibit facial, limb, and cardiac abnormalities. There have been attempts at producing cloned human embryos as sources of embryonic stem cells. Sometimes referred to as cloning for therapeutic purposes, the technique produces stem cells that attempt to remedy detrimental diseases or defects (unlike reproductive cloning, which aims to reproduce an organism). Still, therapeutic cloning efforts have met with resistance because of bioethical considerations.

Basic Techniques to Manipulate Genetic Material (DNA and RNA)

Basic techniques used in genetic material manipulation include extraction, gel electrophoresis, PCR, and blotting methods.

Learning Objectives

Distinguish among the basic techniques used to manipulate DNA and RNA

Key Takeaways

Key Points

  • The first step to study or work with nucleic acids includes the isolation or extraction of DNA or RNA from cells.
  • Gel electrophoresis depends on the negatively-charged ions present on nucleic acids at neutral or basic pH to separate molecules on the basis of size.
  • Specific regions of DNA can be amplified through the use of polymerase chain reaction for further analysis.
  • Southern blotting involves the transfer of DNA to a nylon membrane, while northern blotting is the transfer of RNA to a nylon membrane; these techniques allow samples to be probed for the presence of certain sequences.

Key Terms

  • denaturation: the change of folding structure of a protein (and thus of physical properties) caused by heating, changes in pH, or exposure to certain chemicals
  • electrophoresis: a method for the separation and analysis of large molecules, such as proteins or nucleic acids, by migrating a colloidal solution of them through a gel under the influence of an electric field
  • polymerase chain reaction: a technique in molecular biology for creating multiple copies of DNA from a sample

Basic Techniques to Manipulate Genetic Material (DNA and RNA)

To understand the basic techniques used to work with nucleic acids, remember that nucleic acids are macromolecules made of nucleotides (a sugar, a phosphate, and a nitrogenous base) linked by phosphodiester bonds. The phosphate groups on these molecules each have a net negative charge. An entire set of DNA molecules in the nucleus is called the genome. DNA has two complementary strands linked by hydrogen bonds between the paired bases. The two strands can be separated by exposure to high temperatures (DNA denaturation) and can be reannealed by cooling. The DNA can be replicated by the DNA polymerase enzyme. Unlike DNA, which is located in the nucleus of eukaryotic cells, RNA molecules leave the nucleus. The most common type of RNA that is analyzed is the messenger RNA (mRNA) because it represents the protein -coding genes that are actively expressed.

DNA and RNA Extraction

To study or manipulate nucleic acids, the DNA or RNA must first be isolated or extracted from the cells. This can be done through various techniques. Most nucleic acid extraction techniques involve steps to break open the cell and use enzymatic reactions to destroy all macromolecules that are not desired (such as degradation of unwanted molecules and separation from the DNA sample). Cells are broken using a lysis buffer (a solution that is mostly a detergent); lysis means “to split.” These enzymes break apart lipid molecules in the membranes of the cell and the nucleus. Macromolecules are inactivated using enzymes such as proteases that break down proteins, and ribonucleases (RNAses) that break down RNA. The DNA is then precipitated using alcohol. Human genomic DNA is usually visible as a gelatinous, white mass. Samples can be stored at –80°C for years.

image

DNA Extraction: This diagram shows the basic method used for extraction of DNA.

RNA analysis is performed to study gene expression patterns in cells. RNA is naturally very unstable because RNAses are commonly present in nature and very difficult to inactivate. Similar to DNA, RNA extraction involves the use of various buffers and enzymes to inactivate macromolecules and preserve the RNA.

Gel Electrophoresis

Because nucleic acids are negatively-charged ions at neutral or basic pH in an aqueous environment, they can be mobilized by an electric field. Gel electrophoresis is a technique used to separate molecules on the basis of size using this charge and may be separated as whole chromosomes or fragments. The nucleic acids are loaded into a slot near the negative electrode of a porous gel matrix and pulled toward the positive electrode at the opposite end of the gel. Smaller molecules move through the pores in the gel faster than larger molecules; this difference in the rate of migration separates the fragments on the basis of size. There are molecular-weight standard samples that can be run alongside the molecules to provide a size comparison. Nucleic acids in a gel matrix can be observed using various fluorescent or colored dyes. Distinct nucleic acid fragments appear as bands at specific distances from the top of the gel (the negative electrode end) on the basis of their size.

image

Gel Electrophoresis: Shown are DNA fragments from seven samples run on a gel, stained with a fluorescent dye, and viewed under UV light.

Amplification of Nucleic Acid Fragments by Polymerase Chain Reaction

Polymerase chain reaction (PCR) is a technique used to amplify specific regions of DNA for further analysis. PCR is used for many purposes in laboratories, such as the cloning of gene fragments to analyze genetic diseases, identification of contaminant foreign DNA in a sample, and the amplification of DNA for sequencing. More practical applications include the determination of paternity and detection of genetic diseases.

image

PCR Amplification: Polymerase chain reaction, or PCR, is used to amplify a specific sequence of DNA. Primers—short pieces of DNA complementary to each end of the target sequence—are combined with genomic DNA, Taq polymerase, and deoxynucleotides. Taq polymerase is a DNA polymerase isolated from the thermostable bacterium Thermus aquaticus that is able to withstand the high temperatures used in PCR. Thermus aquaticus grows in the Lower Geyser Basin of Yellowstone National Park. Reverse transcriptase PCR (RT-PCR) is similar to PCR, but cDNA is made from an RNA template before PCR begins.

DNA fragments can also be amplified from an RNA template in a process called reverse transcriptase PCR (RT-PCR). The first step is to recreate the original DNA template strand (called cDNA) by applying DNA nucleotides to the mRNA. This process is called reverse transcription. This requires the presence of an enzyme called reverse transcriptase. After the cDNA is made, regular PCR can be used to amplify it.

Hybridization, Southern Blotting, and Northern Blotting

Nucleic acid samples, such as fragmented genomic DNA and RNA extracts, can be probed for the presence of certain sequences. Short DNA fragments called probes are designed and labeled with radioactive or fluorescent dyes to aid detection. Gel electrophoresis separates the nucleic acid fragments according to their size. The fragments in the gel are then transferred onto a nylon membrane in a procedure called blotting. The nucleic acid fragments that are bound to the surface of the membrane can then be probed with specific radioactively- or fluorescently-labeled probe sequences. When DNA is transferred to a nylon membrane, the technique is called Southern blotting; when RNA is transferred to a nylon membrane, it is called northern blotting. Southern blots are used to detect the presence of certain DNA sequences in a given genome, and northern blots are used to detect gene expression.

image

Blotting Techniques: Southern blotting is used to find a particular sequence in a sample of DNA. DNA fragments are separated on a gel, transferred to a nylon membrane, and incubated with a DNA probe complementary to the sequence of interest. Northern blotting is similar to Southern blotting, but RNA is run on the gel instead of DNA. In western blotting, proteins are run on a gel and detected using antibodies.

Molecular and Cellular Cloning

Molecular cloning reproduces the desired regions or fragments of a genome, enabling the manipulation and study of genes.

Learning Objectives

Describe the process of molecular cloning

Key Takeaways

Key Points

  • Cloning small fragments of a genome allows specific genes, their protein products, and non-coding regions to be studied in isolation.
  • A plasmid, also known as a vector, is a small circular DNA molecule that replicates independently of the chromosomal DNA; it can be used to provide a “folder” in which to insert a desired DNA fragment.
  • Recombinant DNA molecules are plasmids with foreign DNA inserted into them; they are created artificially as they do not occur in nature.
  • Bacteria and yeast naturally produce clones of themselves when they replicate asexually through cellular cloning.

Key Terms

  • recombinant DNA: DNA that has been engineered by splicing together fragments of DNA from multiple species and introduced into the cells of a host
  • molecular cloning: a biological method that creates many identical DNA molecules and directs their replication within a host organism
  • plasmid: a circle of double-stranded DNA that is separate from the chromosomes, which is found in bacteria and protozoa

Molecular Cloning

In general, the word “cloning” means the creation of a perfect replica; however, in biology, the re-creation of a whole organism is referred to as “reproductive cloning.” Long before attempts were made to clone an entire organism, researchers learned how to reproduce desired regions or fragments of the genome, a process that is referred to as molecular cloning.

Cloning small fragments of the genome allows for the manipulation and study of specific genes (and their protein products) or noncoding regions in isolation. A plasmid (also called a vector) is a small circular DNA molecule that replicates independently of the chromosomal DNA. In cloning, the plasmid molecules can be used to provide a “folder” in which to insert a desired DNA fragment. Plasmids are usually introduced into a bacterial host for proliferation. In the bacterial context, the fragment of DNA from the human genome (or the genome of another organism that is being studied) is referred to as foreign DNA (or a transgene) to differentiate it from the DNA of the bacterium, which is called the host DNA.

Plasmids occur naturally in bacterial populations (such as Escherichia coli) and have genes that can contribute favorable traits to the organism such as antibiotic resistance (the ability to be unaffected by antibiotics). Plasmids have been repurposed and engineered as vectors for molecular cloning and the large-scale production of important reagents such as insulin and human growth hormone. An important feature of plasmid vectors is the ease with which a foreign DNA fragment can be introduced via the multiple cloning site (MCS). The MCS is a short DNA sequence containing multiple sites that can be cut with different commonly-available restriction endonucleases. Restriction endonucleases recognize specific DNA sequences and cut them in a predictable manner; they are naturally produced by bacteria as a defense mechanism against foreign DNA. Many restriction endonucleases make staggered cuts in the two strands of DNA, such that the cut ends have a 2- or 4-base single-stranded overhang. Because these overhangs are capable of annealing with complementary overhangs, these are called “sticky ends.” Addition of an enzyme called DNA ligase permanently joins the DNA fragments via phosphodiester bonds. In this way, any DNA fragment generated by restriction endonuclease cleavage can be spliced between the two ends of a plasmid DNA that has been cut with the same restriction endonuclease.

image

Molecular Cloning: This diagram shows the steps involved in molecular cloning, where regions or fragments of a genome are reproduced to allow the study or manipulation of genes and their protein products.

Recombinant DNA Molecules

Plasmids with foreign DNA inserted into them are called recombinant DNA molecules because they are created artificially and do not occur in nature. They are also called chimeric molecules because the origin of different parts of the molecules can be traced back to different species of biological organisms or even to chemical synthesis. Proteins that are expressed from recombinant DNA molecules are called recombinant proteins. Not all recombinant plasmids are capable of expressing genes. The recombinant DNA may need to be moved into a different vector (or host) that is better designed for gene expression. Plasmids may also be engineered to express proteins only when stimulated by certain environmental factors so that scientists can control the expression of the recombinant proteins.

Cellular Cloning

Unicellular organisms, such as bacteria and yeast, naturally produce clones of themselves when they replicate asexually by binary fission; this is known as cellular cloning. The nuclear DNA duplicates by the process of mitosis, which creates an exact replica of the genetic material.

Plasmids as Cloning Vectors

Plasmids can be used as cloning vectors, allowing the insertion of exogenous DNA into a bacterial target.

Learning Objectives

Illustrate how plasmids can be used as cloning vectors

Key Takeaways

Key Points

  • All engineered vectors have an origin of replication, a multi- cloning site, and a selectable marker.
  • Expression vectors (expression constructs) express the transgene in the target cell, and they have a promoter sequence that drives expression of the transgene.
  • Transcription is needed for a plasmid to function, without the proper sequences to transcribe parts of a plasmid it will not be expressed or even maintained in host cells.
  • Vectors can have many additional sequences that can be used for downstream applications—purification of proteins encoded by the plasmid and expressing proteins targeted to be exported or to a certain compartment of the cell.

Key Terms

  • Kozak sequence: a sequence which occurs on eukaryotic mRNA and has the consensus (gcc)gccRccAUGG. The Kozak consensus sequence plays a major role in the initiation of the translation process. The sequence was named after the person who brought it to prominence, Marilyn Kozak.
  • transcription: The synthesis of RNA under the direction of DNA.
  • polyadenylation: The formation of a polyadenylate, especially that of a nucleic acid

Vectors

In molecular biology, a vector is a DNA molecule used as a vehicle to transfer foreign genetic material into another cell. The four major types of vectors are plasmids, viral vectors, cosmids, and artificial chromosomes. All engineered vectors have an origin of replication, a multi-cloning site, and a selectable marker. The vector itself is generally a DNA sequence that consists of an insert (transgene) and a larger sequence, which serves as the “backbone” of the vector. The purpose of a vector that transfers genetic information to another cell is typically to isolate, multiply, or express the insert in the target cell. Vectors called expression vectors (expression constructs) express the transgene in the target cell, and they generally have a promoter sequence that drives expression of the transgene.

Plasmids

Plasmids are double-stranded, generally circular DNA sequences capable of automatically replicating in a host cell. Plasmid vectors minimally consist of the transgene insert and an origin of replication, which allows for semi-independent replication of the plasmid in the host. Modern plasmids generally have many more features, notably a “multiple cloning site”—with nucleotide overhangs for insertion of an insert—and multiple restriction enzyme consensus sites on either side of the insert. Plasmids may be conjugative/transmissible or non-conjugative. Conjugative plasmids mediate DNA transfer through conjugation and therefore spread rapidly among the bacterial cells of a population. Nonconjugative plasmids do not mediate DNA through conjugation.

image

Bacterial Cloning Vector: The pGEX-3x plasmid is a popular cloning vector. The various elements of the plasmid are labelled.

Transcription

Transcription is a necessary component in all vectors. The purpose of a vector is to multiply the insert, although expression vectors also drive the translation of the multiplied insert. Even stable expression is determined by stable transcription, which depends on promoters in the vector. However, expression vectors have a two expression patterns: constitutive (consistent expression) or inducible (expression only under certain conditions or chemicals). Expression is based on different promoter activities, not post-transcriptional activities, meaning these two different types of expression vectors depend on different types of promoters. Expression vectors require translation of the vector’s insert, thus requiring more components than simpler transcription-only vectors.

Expression vectors require sequences that encode for:

  • A polyadenylation tail at the end of the transcribed pre-mRNA: This protects the mRNA from exonucleases and ensures transcriptional and translational termination and stabilizes mRNA production.
  • Minimal UTR length: UTRs contain specific characteristics that may impede transcription or translation, so the shortest UTRs are encoded for in optimal expression vectors.
  • Kozak sequence: a vector should encode for a Kozak sequence in the mRNA, which assembles the ribosome for translation of the mRNA.

The above conditions are necessary for expression vectors in eukaryotes, not prokaryotes.

Modern vectors may encompass additional features besides the transgene insert and a backbone:

  • Promoter: a necessary component for all vectors, used to drive transcription of the vector’s transgene.
  • Genetic markers: Genetic markers for viral vectors allow for confirmation that the vector has integrated with the host genomic DNA.
  • Antibiotic resistance: Vectors with antibiotic-resistance allow for survival of cells that have taken up the vector in growth media containing antibiotics through antibiotic selection.
  • Epitope: A vector containing a sequence for a specific epitope that is incorporated into the expressed protein. Allows for antibody identification of cells expressing the target protein.
  • Reporter genes: Some vectors may contain a reporter gene that allow for identification of plasmid that contains inserted DNA sequence. An example is lacZ-α which codes for the N-terminus fragment of β-galactosidase, an enzyme that digests galactose.
  • Targeting sequence: Expression vectors may include encoding for a targeting sequence in the finished protein that directs the expressed protein to a specific organelle in the cell or specific location such as the periplasmic space of bacteria.
  • Protein purification tags: Some expression vectors include proteins or peptide sequences that allows for easier purification of the expressed protein. Examples include polyhistidine-tag, glutathione-S-transferase, and maltose binding protein. Some of these tags may also allow for increased solubility of the target protein. The target protein is fused to the protein tag, but a protease cleavage site positioned in the polypeptide linker region between the protein and the tag allows the tag to be removed later.