7.12A: Recombinant DNA Technology - Biology

7.12A: Recombinant DNA Technology - Biology

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


Show some of the methods and uses of recombinant DNA

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.

Ensuring Safety in Biotechnology

9.2.3 Safety Issues in the Use of Recombinant DNA Technology in Medicine

Recombinant DNA technology has contributed to health care in two important ways: production of pharmaceutically important proteins (biopharmaceuticals) and gene therapy for replacement of defective genes. Biopharmaceuticals

Recombinant DNA technology has been effectively used to produce various human proteins in microorganisms, such as insulin and growth hormone, used in the treatment of diseases (see Chapter 4 : Recombinant DNA Technology and Genetically Modified Organisms). Unlike chemically synthesized drugs, these are biomacromolecules—primarily endogenous proteins, and present a variety of special considerations and concerns:

whether the molecule produced through rDNA technology is biologically equivalent to the naturally occurring one

as these are mostly proteins, will they result in immunogenic reactions that would limit their usefulness.

Testing of these compounds presents unique problems. For instance, since they are endogenously produced, assessing pharmacokinetics and metabolism is difficult. Also, since they are available only in small quantities, traditional testing protocols that involve progressively increasing dosages until adverse effects occur, may not be possible. For conventional pharmaceutical safety assessment, the compound is to be tested separately in at least two mammalian species of which one must be a nonrodent. With substances with specific activity in humans, the evaluation in rodent and other model species may not be appropriate. Differences in immunological sensitivities in animal and human systems can have disastrous effects as was seen in the TeGenero trial for testing an antibody TGN1412 intended to treat rheumatoid arthritis and B-cell chronic lymphocyte leukemia (see Chapter 5 : Relevance of Ethics in Biotechnology). Gene therapy

Gene therapy aims to treat/cure/prevent disease by replacing a defective gene with a normal one using recombinant DNA technology . Most human clinical trials in gene therapy are still in the research stage with only over 400 trials conducted in about 3000 patients in order to treat for single gene disorders, cancers, and AIDS. Scientists agree that this is the most powerful application of rDNA technology, but have been cautious in its application due to associated risks as exemplified by the case of Jesse Gelsinger (see Chapter 4 : Recombinant DNA Technology and Genetically Modified Organisms). Ensuring safety of patients in clinical trials has led to the development of better risk assessment in clinical trials (see Chapter 10 : Risk Analysis).

Safety issues in recombinant DNA technology include: •

“Gene pollution” of the environment resulting in “superweeds,” antibiotic-resistant microbes

Health effects of foods from GMOs

Allergenicity/adverse immune reactions/effectiveness of pharmaceutical compounds produced using rDNA technology

Recombinant DNA Technology: Importance, Concepts and Application

In this article we will discuss about the Recombinant DNA Technology:- 1. Introduction to Recombinant DNA Technology 2. Biomedical Importance of Recombinant DNA Technology 3. Concept 4. Some Practical Applications.

Introduction to Recombinant DNA Technology:

a. Recombinant DNA technology is better referred to as genetic engineering.

b. Much has been learned about the diseases from the study of affected proteins, but this mechanism cannot be applied where the specific genetic defect is unknown. This new technology overcoming these limitations will approach directly to the DNA molecule for information.

Biomedical Importance of Recombinant DNA Technology:

a. It is helpful to give clear idea regarding the molecular basis of a number of dis­eases (e.g., familial hypercholesterolemia, sickle cell disease, the thalassemia, cystic fibrosis, Huntington’s chorea).

b. Using this technology, a large quantity of human proteins can be produced for therapy.

c. By its aid proteins for vaccines (e.g., hepa­titis B) and for diagnostic tests (e.g., AIDS test) can be obtained.

d. This technology is utilized to diagnose existing diseases and predict the risk of developing a given disease.

e. Gene therapy for sickle cell disease, the thalassemia’s, adenosine deaminase defi­ciency, and other diseases may be devised.

Concept used in Recombinant DNA Technology:

Isolation and manipulation of DNA is the object of recombinant DNA research. This requires several techniques and reagents.

Restriction Enzymes:

a. Some endonucleases that cut DNA at spe­cific DNA sequences within the molecule are a key tool in recombinant DNA re­search. These enzymes were originally said to be restriction enzymes. More than 200 defensive enzymes protect the host bacte­rial DNA from foreign organism (prima­rily infective phages).

They are only present in cells that also have a compan­ion enzyme that methylate’s the host DNA giving it an unsuitable substrate for di­gestion by the restriction enzyme.

b. The restriction enzymes are named vide the bacterium from which they are isolated (e.g., Eco RI from Escherichia Coli, Bam HI from bacillus amyloliquefaciens).

c. Each enzyme recognizes and cleaves a specific double-stranded DNA sequence. These DNA cut result in blunt ends or over­lapping (sticky) ends (Bam HI) (Fig. 24.1), depending on the mechanism used by the enzyme. Sticky ends are particularly use­ful in constructing hybrid or chimeric DNA molecules.

d. In case the nucleotides are distributed ran­domly in a given DNA molecule, one can easily calculate how frequently a given enzyme could cut a length of DNA.

e. For each position in the DNA molecule there are 4 possibilities (A,C, G or T) therefore, a restriction enzyme that recog­nizes a 4-bp sequence will cut, on aver­age, once every (4 4 ), whereas another en­zyme that recognizes a 6-bp sequence will cut once every (4 6 ).

f. When DNA is digested with a given en­zyme, the ends of all the fragments will have the same DNA sequence. The frag­ments produced can be isolated by elec­trophoresis.

Preparation of Chimeric DNA Molecules:

a. Sticky ends of a vector may reconnect with themselves with no gain of DNA. These ends of fragments can also anneal so that tandem heterogeneous inserts form. These end sites may not be available or in a con­venient position.

b. To overcome the above problems, an en­zyme that generates blunt ends is used and new ends are added using the enzyme ter­minal transferase.

c. If poly d(G) is added to the 3′ ends of the vector and poly d(C) is added to the 3′ ends of the foreign DNA, the two molecules can only anneal to each other and thus overcome the above problem. This proce­dure, called homopolymer tailing, also generates an Sma I restriction site.

d. Sometimes, synthetic oligonucleotide linkers with a convenient restriction en­zyme sequence are ligated to the blunt- ended DNA. This technique has the ad­vantage of joining together any pairs of ends. The dis-advantages are that there is no control over the orientation of inser­tion of the number of molecules annealed together.

Some Practical Applications on Recombinant DNA Technology:

a. Specific genes to distinct chromosomes are localized by this technique and thus to define a map of the human genome. This is already producing useful informa­tion in the definition of human disease.

b. Somatic cell hybridization and in situ hybridization are two techniques used to accomplish this.

c. In hybridization, the simpler and more direct procedure, a radioactive probe is added to a metaphase spread of chromo­somes on a glass slide. The exact area of hybridization is localized by layering photographic emulsion over the slide and, after exposure lining up the grains with some histologic identification of the chro­mosome. Some of the human genes are lo­calized by this technique.

d. Genes that code for proteins with similar functions can be located on separate chro­mosomes.

e. Genes that form part of a family can also be on separate chromosomes (Growth hor­mone and prolactin).

f. The genes involved in many hereditary disorders known to be due to specific pro­tein deficiencies, including X chromo­some-linked conditions, are really located at specific sites.

Protein Production:

a. This technology has two prominent mer­its:

(i) It can supply large amounts of mate­rials that could not be obtained by conventional purification methods.

(ii) It can provide human material (e.g., in­sulin, growth hormone).

b. Although the primary aim is to supply prod­ucts, generally proteins, for treatment (In­sulin) and diagnosis (AIDS test) of human and other animal disease and for disease prevention (hepatitis B vaccine), there are other real and potential commercial ap­plications, especially in agriculture.


The goal of this experiment is to study about-Recombinant DNA Technology In Today&rsquos Medicine.

Genetic Engineering

Genetic Engineering plays a very important role, not only in scientific research, but also in the diagnosis and treatment of disease. Recombinant DNA is a tool in understanding the structure, function, and regulation of genes and their products.

The objectives of Recombinant DNA technology include:

&ndash Re-expressing genes in other hosts or organisms

 These steps permit scientists and clinicians to:

&ndash Identify new genes and the proteins they encode

&ndash To correct endogenous genetic defects

&ndash To manufacture large quantities of specific gene products such as hormones, vaccines, and other biological agents of medical interest

 Genetic engineering produces proteins that offer advantages over proteins isolated from other biological sources. These advantages include:

Steps in Synthesizing a Recombinant Protein

 Recombinant technology begins with the isolation of a gene of interest. The gene is then inserted into a vector and cloned. A vector is a piece of DNA that is capable of independent growth commonly used vectors are bacterial plasmids and viral phages. The gene of interest (foreign DNA) is integrated into the plasmid or phage, and this is referred to as recombinant DNA.

 Before introducing the vector containing the foreign DNA into host cells to express the protein, it must be cloned. Cloning is necessary to produce numerous copies of the DNA since the initial supply is inadequate to insert into host cells.

 Once the vector is isolated in large quantities, it can be introduced into the desired host cells such as mammalian, yeast, or special bacterial cells. The host cells will then synthesize the foreign protein from the recombinant DNA. When the cells are grown in vast quantities, the foreign or recombinant protein can be isolated and purified in large amounts.

 Recombinant DNA technology is not only an important tool in scientific research, but has also resulted in enormous progress in the diagnosis and treatment of certain diseases and genetic disorders in many areas of medicine.

Genetic engineering has permitted

Identification of mutations:

People may be tested for the presence of mutated proteins that may be involved in the progression of breast cancer, retino-blastoma, and neurofibromatosis

Diagnosis of affected and carrier states for hereditary diseases:

Tests exist to determine if people are carriers of the cystic fibrosis gene, the Huntington&rsquos disease gene, the Tay-Sachs disease gene, or the Duchenne muscular dystrophy gene.

Mapping of human genes on chromosomes:

Scientists are able to link mutations and disease states to specific sites on chromosomes.

Transferring genes from one organism to another:

People suffering from cystic fibrosis, rheumatoid arthritis, vascular disease, and certain cancers may now benefit from the progress made in gene therapy.

Isolation and alteration of genes:

Once gene modification becomes successful, alteration of genes to produce a more functional protein than the endogenous protein may become possible, opening up the route of gene therapy.

Performing structure and function analyses on proteins:

Researchers may now employ rational drug design to synthesize drug compounds that will be efficacious and selective in treating disease.

Isolation of large quantities of pure protein:

Insulin, growth hormone, follicle-stimulating hormone, as well as other proteins, are now available as recombinant products. Physicians will no longer have to rely on biological products of low purity and specific activity from inconsistent batch preparations to treat their patients

Recombinant DNA Technique

 Restriction enzymes used to cut out insulin gene and to cut a bacterial (E. coli) plasmid at the same &ldquosticky ends&rdquo

 Mutant strains of E. coli used to avoid bacteria attacking &ldquoforeign&rdquo genes

 Insert insulin gene next to E. coli

B-galactosidase gene which controls transcription

 Bacterial cells replicate and make copies of insulin gene

 Insulin protein is purified (B-galactosidase removed)

 Chains are mixed and disulfide bridges form

 Yeast cells provide a sterile growth medium

 Final product is Humulin - chemically identical to human insulin

Plasmid Polylinkers and Marker Genes for Blue-White Screening

 A vector usually contains a sequence (polylinker) which can recognize several restriction enzymes so that the vector can be used for cloning a variety of DNA samples.

 Colonies with recombinant plasmids are white, and colonies with nonrecombinant plasmids are blue.

 Resistant to ampicillin, has (amprgene)

 Contains portion of the lac operon which codes for beta-galactosidase.

 X-gal is a substrate of beta-galactosidase and turns blue in the presence of functional beta-galactosidase is added to the medium.

 Insertion of foreign DNA into the polylinker disrupts the lac operon, beta-galactosidase becomes non-functional and the colonies fail to turn blue, but appear white.

Bacterial Artificial Chromosomes(BACS) And Yeast Artificial Chromosomes(YACS)

 BACs can hold up to 300 kbs.

 The F factor of E.coli is capable of handling large segments of DNA.

 Recombinant BACs are introduced into E.coli by electroportation( a brief high-voltage current). Once in the cell, the rBAC replicates like an F factor.

 Has a set of regulatory genes, OriS, and repE which control F-factor replication, and parA and parB which limit the number of copies to one or two.

 A chloramphenicol resistance gene, and a cloning segment.

 YACs can hold up to 500 kbs.

 YACs are designed to replicate as plasmids in bacteria when no foreign DNA is present. Once a fragment is inserted, YACs are transferred to cells, they then replicate as eukaryotic chromosomes.

 YACs contain: a yeast centromere, two yeast telomeres, a bacterial origin of replication, and bacterial selectable markers.

 YAC plasmid ----> Yeast chromosome

DNA is inserted to a unique restriction site, and cleaves the plasmid with another restriction endonuclease that removes a fragment of DNA and causes the YAC to become linear. Once in the cell, the rYAC replicates as a chromosome, also replicating the foreign DNA.

Why is a Lentivirus necessary?

Lentiviruses can introduce a gene of interest into cells that do not divide &ndash simple retroviruses cannot.

This ability makes them ideal for a delivery system because most of our cells, like hemopoietic stem cells, do not divide.

Why use HIV?

A genetically stripped down amalgam of HIV components can be fashioned with a molecular switch system that turns them off in response to a common antibiotic.

This type of control allows doctors to control gene expression in people who are treated with gene therapy - If something goes wrong, the expression can be turned off.

Adenoviruses are often used as a vector in gene therapy research but they do not have the capacity to integrate their genome into the hosts genome.

The advantage to using a retrovirus is that you don&rsquot lose the genomic sequence that is incorporated into the host DNA following cell division.

Pacific Northwest National Laboratory, Richland, Washington

A. Massey & Associates, Chapel Hill, North Carolina

Pacific Northwest National Laboratory, Richland, Washington

A. Massey & Associates, Chapel Hill, North Carolina

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This chapter looks at some of the tools scientists use in carrying out research of recombinant DNA. It looks at some of the enzymes and other fundamental tools for working with DNA and cells that are used to manipulate and analyze DNA, including determining its sequence to clone DNA and to analyze proteins. The chapter discusses ways in which these tools are used to accomplish specific goals, such as finding genes, analyzing genotypes, generating DNA fingerprints, and genetically engineering both plants and animals. The study of model organisms and the sequencing of their genomes are paying off handsomely in this arena. If the genotypes or genomes of different species are compared, one can ask how closely related those species might be and draw conclusions about the course of evolution. Shifting the focus to the level of individuals, the genotypes can be compared and conclusions can be drawn about whether the individuals are related or whether two samples came from the same individual. The goal of genetic engineering is not to manipulate an organism's DNA per se but to change something about the proteins produced in that organism: to cause it to produce a new protein, to stop producing an old protein, to produce more or less of a protein, and so on.

Recombinant DNA Technology

All organisms on Earth evolved from a common ancestor, so all organisms use DNA as their molecule of heredity. At the chemical level, DNA is the same whether it is taken from a microscopic bacterium or a blue whale. As a result, DNA from different organisms can be “cut and pasted” together, resulting in “recombinant DNA”. The first recombinant DNA molecule was produced in 1972 by Stanford researcher Paul Berg. Berg joined together DNA fragments from two different viruses with the help of particular enzymes: restriction enzymes and ligase. Restriction enzymes (such as EcoR1 in the figure below) are like “molecular scissors” that cut DNA at specific sequences. If the DNA from the different sources is cut with the same restriction enzyme, the cut ends can be joined together and then sealed into a continuous DNA strand by the enzyme ligase. In 1973, the first organism to contain recombinant DNA was engineered by Herb Boyer (UCSF) and Stanley Cohen (Stanford University). Together they introduced an antibiotic resistance gene into E.coli bacteria. Notably, they also produced bacteria that contained genes from the toad Xenopus laevis , which showed DNA from very different species could be spliced together. Paul Berg was awarded the 1980 Nobel Prize in Chemistry “for his fundamental studies of the biochemistry of nucleic acids, with particular regard to recombinant-DNA”.

The production of recombinant DNA involves cutting two different pieces of DNA with the same restriction enzyme and then ligating (“glueing”) the pieces together. Image courtesy of Wikimedia Commons

The ability to cut, paste, and copy molecules of DNA was not only a watershed moment for scientific research but spawned an entire industry built on genetic engineering. Genetech, the first biotechnology company, was founded by Herb Boyer in 1976. By 1982, the FDA approved Genetech’s first successful product, a synthetic form of human insulin produced by bacteria that were engineered to contain the insulin gene.

Today recombinant DNA technology is used extensively in research laboratories worldwide to explore myriad questions about gene structure, function, expression pattern, regulation, and much more. One widely used application involves genetically engineering “knock-out” animals (typically mice) to contain a non-functional form of a particular gene of interest. The goal of such experiments is to determine gene function by analyzing the consequences of the missing gene. While knockout mice are generated to answer questions in many different fields, they are particularly useful in developmental biology and have led to an understanding of some of the essential genes involved in the development of an organism from a single fertilized egg.

Recombinant DNA techniques are also a cornerstone of the biotechnology industry. One example is the generation of genetically engineered plants to produce an insect toxin called Bt toxin. The Bt gene is derived from a bacterium called Bacillus thuringiensis and produces a toxin that disrupts gut function in the larvae (caterpillars) of certain insects that are crop pests. The gene that produces Bt toxin is introduced into such plants by recombinant DNA technology, and results in the selective killing of crop-feeding insects. This development has had a major economic impact and reduced the expenses of pesticides used per year and has increased the longevity and success of several crops.

CLICK HERE to learn more about transgenic organisms
CLICK HERE to learn more about synthetic biology
CLICK HERE to learn more about cloning

CLICK HERE for a case study that addresses one of the biosafety concerns of recombinant DNA technology

Pacific Northwest National Laboratory, Richland, Washington

A. Massey & Associates, Chapel Hill, North Carolina

Pacific Northwest National Laboratory, Richland, Washington

A. Massey & Associates, Chapel Hill, North Carolina


This chapter looks at some of the tools scientists use in carrying out research of recombinant DNA. It looks at some of the enzymes and other fundamental tools for working with DNA and cells that are used to manipulate and analyze DNA, including determining its sequence to clone DNA and to analyze proteins. The chapter discusses ways in which these tools are used to accomplish specific goals, such as finding genes, analyzing genotypes, generating DNA fingerprints, and genetically engineering both plants and animals. The study of model organisms and the sequencing of their genomes are paying off handsomely in this arena. If the genotypes or genomes of different species are compared, one can ask how closely related those species might be and draw conclusions about the course of evolution. Shifting the focus to the level of individuals, the genotypes can be compared and conclusions can be drawn about whether the individuals are related or whether two samples came from the same individual. The goal of genetic engineering is not to manipulate an organism's DNA per se but to change something about the proteins produced in that organism: to cause it to produce a new protein, to stop producing an old protein, to produce more or less of a protein, and so on.

Various commercially important chemicals can be produced more efficiently by utilizing the methods of rec DNA technology. A few of them are the alcohols and alcoholic beverages obtained through fermentation organic acids like citric acid, acetic acid, etc. and vitamins produced by microorganisms.

As we know that the enzymes are encoded by genes, so if there are changes in a gene then definitely the enzyme structure also changes. Enzyme engineering utilizes the same fact and can be explained as the modification of an enzyme structure by inducing alterations in the genes which encode for that particular enzyme.

Recombinant DNA

Many of our drugs, much of our food, and even our clothing are now produced using recombinant DNA technology. Instead of depending on random mutation, and either natural or artificial selection, we now have the ability to directly manipulate the genes of organisms to create new proteins and new capabilities in our domesticated bacteria, fungi, plants and animals.

Molecular cloning

Molecular biologists coined the term “molecular cloning” to describe the process of selectively replicating a chosen segment of DNA. The cloned DNA segment may be replicated within a cell, using “recombinant DNA” technology, or in a test tube, using the polymerase chain reaction (PCR).

Recombinant DNA technology leads to genetically modified organisms (GMOs). Recombinant DNA requires 3 key molecular tools:

  1. Cutting DNA at specific sites – most often performed by enzymes called restriction endonucleases (restriction enzymes). Restriction enzymes often make staggered cuts at specific 4, 6, or 8-bp palindromic sequences in duplex DNA, leaving characteristic “sticky ends” that can anneal to each other via hydrogen bonding between complementary bases on the single-stranded overhangs.
  2. Ligating DNA fragments with an enzyme called DNA ligase. DNA ligase, the same enzyme used during cellular DNA replication to knit together Okazaki fragments, creates covalent phosphodiester bonds between any two DNA fragments that have been cut by the same restriction enzyme, or have the same compatible “sticky ends”.
  3. A “vector”, such as a plasmid, that can be used to insert a new segment of DNA via restriction enzyme cutting and ligation. The plasmid containing the inserted DNA segment will replicate in host cells.

The alternative to using recombinant plasmids is to directly copy and amplify a specific DNA segment using PCR. PCR requires a pair of primers that correspond to the ends of the desired DNA segment.

Even random DNA segments, where the base sequences are unknown, may be amplified by ligating adapter primers, short synthetic DNA segments of known sequence, to the ends of the target DNA molecules.

Cloning eukaryotic genes

Molecular cloning of eukaryotic genes is often either unfeasible or undesirable, or both, because they contain numerous, large introns. Plasmid vectors have a practical size limit of less than 10 kilo-base pairs (kbp), and PCR is also difficult beyond about 10 kb.

The mRNA, lacking introns, is a compact version of a eukaryotic gene that retains all of the protein coding information. The enzyme reverse transcriptase can be used, along with an oligo-dT primer that is complementary to the polyA tail, to synthesize a complementary DNA (cDNA) molecule. The cDNA can then be cloned into a plasmid or amplified by PCR by ligating adapters that contain restriction endonuclease cleavage sites or PCR primer sequences.

A cDNA is synthesized from mRNA using reverse transcriptase and olig-dT primers. The reverse transcriptase will also degrade the mRNA from the cDNA:mRNA hybrid molecule. DNA polymerase uses the mRNA fragments or random primers to synthesize the 2nd strand of the cDNA molecule. Adapters with restriction endonuclease sites or PCR primer sequences can be ligated to the ends of the completed cDNA to facilitate cloning into plasmids or amplification by PCR. Original illustration by J. Choi

Expressing cloned genes: genetically modified organisms

A map of pUC18, Figure 1 from Bensasson et al. 2004 Heredity 92:483A genetically modified organism (GMO) is any organisms that has been manipulated so it carries new genetic material, from either a different species or synthesized in the laboratory. The point of creating GMOs is usually to alter their traits, most often so they express a new gene.

Expression of foreign genes in bacteria

Plasmid vectors for cloning and expression in bacteria (see pUC18 map above) must have

  • An origin of DNA replication (ori) that directs their replication in the host cell
  • restriction endonuclease sites (polylinker) that occur just once on the vector, for insertion of cloned DNA segments
  • a selectable marker gene, such as antibiotic resistance (bla encodes beta-lactamase for ampicillin resistance), so cells that do not contain the plasmid can be eliminated
  • a way to distinguish cells that have the original plasmid from cells that have a recombinant plasmid.
  • a promoter to drive transcription (and translation) of the inserted foreign gene

The last feature is important because the ligation of plasmid and foreign DNA segments favors the plasmid ends re-ligating without a foreign DNA insert, resulting in the original, “empty” plasmid with no foreign DNA insert. Plasmid vectors therefore have the cloning site within a second antibiotic resistance gene or within the lacZ gene (encodes beta-galactosidase). Insertion of a foreign DNA segment will disrupt the gene. Colonies of E. coli cells that have empty plasmids (no inserted foreign DNA) have an intact lacZ gene, produce functional beta-galactosidase, and cleave a colorless dye called X-gal to release the insoluble blue dye X, and turn blue. E. coli cells that have plasmids with foreign DNA inserts make no beta-galactosidase, and are unable to cleave X-gal. These colonies stay white. Blue colonies are discarded, and white colonies are picked for further testing.

Cloning into the 5′ end of the lacZ gene also means that the E. coli cell can express a protein encoded by the inserted DNA. The lac promoter provides a means to regulate transcription, and protein coding sequences in the inserted DNA can be expressed as a fusion protein, containing the first few amino acids of the E. coli beta-galactosidase gene, and any amino acids encoded in the same reading frame by the inserted DNA sequence.

Expression of foreign genes in eukaryotes

Vectors for expression of foreign genes in eukaryotic cells must provide appropriate eukaryotic promoters upstream of the cloning site, for transcription by the eukaryotic host cell, as well as downstream polyadenylation and transcription termination signals. For single-celled organisms such as yeast, and cultured cells, bacterial plasmids containing foreign genes can be transformed into the cells. The plasmid DNA gets into the nucleus, and inserts into random locations in the host cell’s chromosomes. For multicellular organisms, the delivery of genes into the cells of the organism poses special challenges and requires special vectors and delivery methods. We describe these challenges for one application, human gene therapy, in the next section.

Gene therapy

Gene therapy poses a special challenge in delivering recombinant DNA into host cells. Recombinant DNA technology can readily clone a functional copy of a defective gene and insert it into a vector with the correct regulatory sequences. But how can we deliver this functional gene into the cells of a person who has already been born? The most promising techniques use viruses. Viruses evolved to be highly efficient at delivering their own genetic information into host cells. Replacing the viral replication genes with a therapeutic human gene eliminates the ability of the virus to replicate, while co-opting the viral infection mechanism to deliver the therapeutic gene into the nuclei of host cells.

Even then, only a small percentage of cells are infected and repaired (remember these therapeutic viruses can’t replicate to infect other cells). Moreover, benefits of viral gene therapy are short-lived, as the “repaired” cells age, die and are replaced by genetically unmodified cells.

A promising solution to these challenges is to find and genetically modify stem cells, those cells that will continue to divide and replenish the body’s cells for the rest of the patient’s life. Genetically modified stem cells can be returned to the patient’s body and have the potential to supply and replenish genetically modified blood cells and tissues for the rest of the patient’s life.

Genome editing

One technology developed in recent years, and being widely adopted in research labs around the world, is CRISPR-Cas9 technology, and variants. CRISPR stands for C lustered R egularly I nterspersed S hort P alindromic R epeats. Cas9 is a protein enzyme that binds short RNAs made from CRISPR genes to recognize and cleave DNA sequences that match the CRISPR RNAs. This technology enables researchers to delete, add, or replace particular bits of DNA in a cell. Human genome editing may be less controversial than human genetic modification, because no non-human DNA is added.

Here is a TED talk video by Jennifer Doudna, one of the developers of CRISPR technology and a winner of the 202 Nobel Prize for Chemistry:

In essence, Cas9 is a protein that cuts DNA. Whereas restriction endonucleases cut DNA at fixed sites, Cas9 is programmable. Cas9 targets the DNA site to be cut by using a short guide RNA (sgRNA). Cas9 binds the sgRNA, and cuts DNA wherever the sgRNA binds to a complementary DNA sequence. So in any organism where the genome sequence is known, scientists can make an sgRNA to target a particular DNA sequence in the genome, and cut it. After Cas9 cuts the DNA to create a double-strand DNA break, the cell’s DNA repair system will trim the broken ends and ligate them together, often creating small deletions as a result of the trimming. If a homologous DNA sequence is available (matches the sequences around the cut ends), the cell’s DNA repair system uses the matching DNA as a template to repair the break in the DNA. This homology-dependent repair system copies the sequence information in the DNA template as it joins the broken ends together. By providing Cas9 protein, sgRNA, and a homologous template DNA that includes a desired change, scientists have successfully made precise changes in genomes of many kinds of cells and organisms, including cultured human cells.

Cas9 protein and sgRNA bound to target DNA, based on structure by Anders et al 2014 Nature. CC-BY-SA by Cas9 Wiki project.

After cleavage by Cas9, the cell’s own DNA break repair system will use a homologous DNA sequence to result in replacement of the targeted gene, or use a non-homologous end joining that causes small deletion mutations. Modified by JChoi from CC-BY-SA image by Carroll, D. Genome engineering with zinc-finger nucleases. Genetics Society of America, 2011, 188(4), pp 773-782.

Put it all together:

In class we will discuss how these concepts are applied to current gene therapy methods undergoing research and development.

Dr. Choi’s lecture video on recombinant DNA technology (in one 39-min chunk, until I find time to redo this in multiple short segments):

To insert a mammalian gene into a prokaryotic cell, two basic requirements must be met.

  • First, researchers must isolate the target mammalian gene from the genome as a whole.
  • Second, the researchers must find a way to ensure that the prokaryotic cell can express the mammalian gene correctly.

Creating and Isolating the Target Gene

The eukaryotic chromosome and selected bacterial plasmids to be used as a cloning vector are treated with a restriction endonuclease.

When the eukaryotic DNA fragments are combined with the broken plasmids, some of the plasmids recombine with eukaryotic DNA.

The plasmids are then returned to the host bacteria by simply culturing both in solution so that some of the bacteria will take up the plasmids.

However, many of the plasmids will not contain recombinant DNA of those that do, only a small portion will contain the target mammalian gene.

Therefore, the next step is to isolate bacterial colonies that contain the recombinant plasmids incorporating the target gene.

This step involves two stages of screening:

Stage 1: Identify the bacterial colonies that contain recombinant plasmids.

Only a portion of the bacteria will take up recombinant plasmids.

To identify those that do, researchers typically use plasmids carrying a particular genetic marker — that is, a trait that is easily identified.

Stage 2: Identify the bacteria containing the desired gene.

When the mammalian DNA is broken with an endonuclease, the result is likely to be hundreds or thousands of fragments. Of these, only a small fraction will contain the target gene.

As a result, another step is required to find those bacteria that contain a plasmid that includes the right gene.

Identifying these bacteria involves the use of a nucleic acid probe in a technique called nucleic acid hybridization.

If at least part of the nucleic acid sequence of the gene is known, this information can be used to construct a probe made of RNA or single-stranded DNA. The probe consists of a nucleic acid sequence complementary to the known gene sequence, along with a radioactive or fluorescent tag.

To employ the probe, DNA from each bacterial colony is first heated to separate its two strands and then mixed with a solution containing the nucleic acid probe.

The probe forms a base pair with its complementary sequence, making it possible for researchers to locate the tag to determine which bacterial colony contains the desired gene.

Once the colony has been identified, it can be cultured to produce the gene product.

Expressing Eukaryotic Genes in Prokaryote Vectors

First, the promoter sequence of a eukaryotic gene will not be recognized by the prokaryotic form of RNA polymerase.

To overcome this problem, researchers have developed a particular type of plasmid called an expression vector.

An expression vector is a plasmid that contains a prokaryotic promoter sequence just ahead of a restriction enzyme target site. Thus, when recombination occurs, the inserted DNA sequence will lie close to the bacterial promoter. The host cell then recognizes the promoter and transcribes the gene.

Second, a prokaryote does not contain the snRNA or spliceosomes necessary to remove introns from a eukaryotic pre-mRNA transcript.

This means that the mRNA transcript in a prokaryote will contain both coding and non-coding sequences, both of which will be translated by the cell.

The solution to this problem has been to develop artificial eukaryotic genes that do not contain introns.

Researchers first isolate finished mRNA from the cytoplasm of an eukaryotic cell.

The mRNA is then placed in a solution with an enzyme called reverse transcriptase, which creates a DNA strand complementary to the mRNA strand.

This DNA strand is then isolated and added to a solution containing DNA polymerase, which synthesizes another complementary DNA strand.

The result is a double-stranded molecule of DNA containing only the coding portions of the eukaryotic gene. This synthetic molecule is called copy DNA or cDNA.

Another solution to both of these problems is to use eukaryotic cells as cloning vectors.

Yeast cells are often used for this purpose, since they can be cultured easily. Some yeast cells also contain plasmids, so similar techniques can be used to insert recombinant DNA into the cloning vector.

Inserting DNA into Plant or Animal Vectors

In some cases, only plant or animal cells will contain all the enzymes necessary to correctly manufacture a desired protein. Such cells can be grown in cultures to serve as cloning vectors.

However, because these cells are more difficult to culture, it is harder to insert foreign DNA into them.

To get around this apparent barrier and place foreign genes into eukaryotic genomes, biologists have developed several methods.

Amplification of DNA by the Polymerase Chain Reaction

Molecular cloning allows individual DNA fragments to be propagated in bacteria and isolated in large amounts. An alternative method to isolating large amounts of a single DNA molecule is the polymerase chain reaction (PCR), which was developed by Kary Mullis in 1988. Provided that some sequence of the DNA molecule is known, PCR can achieve a striking amplification of DNA via reactions carried out entirely in vitro. Essentially, DNA polymerase is used for repeated replication of a defined segment of DNA. The number of DNA molecules increases exponentially, doubling with each round of replication, so a substantial quantity of DNA can be obtained from a small number of initial template copies. For example, a single DNA molecule amplified through 30 cycles of replication would theoretically yield 2 30 (approximately 1 billion) progeny molecules. Single DNA molecules can thus be amplified to yield readily detectable quantities of DNA that can be isolated by molecular cloning or further analyzed directly by restriction endonuclease digestion or nucleotide sequencing.

The general procedure for PCR amplification of DNA is illustrated in Figure 3.27. The starting material can be either a cloned DNA fragment or a mixture of DNA molecules𠅏or example, total DNA from human cells. A specific region of DNA can be amplified from such a mixture, provided that the nucleotide sequence surrounding the region is known so that primers can be designed to initiate DNA synthesis at the desired point. Such primers are usually chemically synthesized oligonucleotides containing 15 to 20 bases of DNA. Two primers are used to initiate DNA synthesis in opposite directions from complementary DNA strands. The reaction is started by heating the template DNA to a high temperature (e.g., 95ଌ) so that the two strands separate. The temperature is then lowered to allow the primers to pair with their complementary sequences on the template strands. DNA polymerase then uses the primers to synthesize a new strand complementary to each template. Thus in one cycle of amplification, two new DNA molecules are synthesized from one template molecule. The process can be repeated multiple times, with a twofold increase in DNA molecules resulting from each round of replication.

Figure 3.27

Amplification of DNA by PCR. The region of DNA to be amplified is flanked by two sequences used to prime DNA synthesis. The starting double-stranded DNA is heated to separate the strands and then cooled to allow primers (usually oligonucleotides of 15 (more. )

The multiple cycles of heating and cooling involved in PCR are performed by programmable heating blocks called thermocyclers. The DNA polymerases used in these reactions are heat-stable enzymes from bacteria such as Thermus aquaticus, which lives in hot springs at temperatures of about 75ଌ. These polymerases are stable even at the high temperatures used to separate the strands of double-stranded DNA, so PCR amplification can be performed rapidly and automatically. RNA sequences can also be amplified by this method if reverse transcriptase is used to synthesize a cDNA copy prior to PCR amplification.

If enough of the sequence of a gene is known that primers can be specified, PCR amplification provides an extremely powerful method of obtaining readily detectable and manipulable amounts of DNA from starting material that may contain only a few molecules of the desired DNA sequence in a complex mixture of other molecules. For example, defined DNA sequences of up to several kilobases can be readily amplified from total genomic DNA, or a single cDNA can be amplified from total cell RNA. These amplified DNA segments can then be further manipulated or analyzed, for example, to detect mutations within a gene of interest. PCR is thus a powerful addition to the repertoire of recombinant DNA techniques. Its power is particularly apparent in applications such as the diagnosis of inherited diseases, studies of gene expression during development, and forensic medicine.