Clear and concise, this easy-to-use book offers an introductory course in the language of gene cloning. Part I outlines the essentials of biology and genetic processes, emphasizing those relevant to cloning, while Part II describes common techniques and approaches for microbial, plant, and mammalian systems.
This revised edition includes updated information, a tutorial on reading a sequence, new materials for animal cloning, and rewritten chapters on DNA typing and human gene therapy.
What is Gene Cloning?
We may picture Dolly the sheep or other cloned animals when we think of cloning. However, when molecular biologists talk about cloning something, they are almost always talking about copying a gene or small piece of DNA. In fact, scientists have been cloning genes and other small pieces of DNA for years, long before any attempts were made to clone entire organisms.
Cloning is the creation of an identical copy. A clone can be a gene, cell, animal, or plant in biology. A cloned cell can produce the same protein as the original cell but cannot reproduce itself because multicellular organisms reproduce sexually and require the contribution of DNA from both parents.
Scientists first extract the DNA from the target organism and cut it into gene-size fragments with restriction enzymes to make a clone. The fragments are then inserted into vectors that can transfer the recombinant DNA to host cells. Vectors can be bacterial plasmids, phages, or bacteriophages, or “naked” DNA molecules covalently linked to a cloning enzyme (polymerase chain reaction, or PCR).
Once the DNA is inside a host cell, it can begin to replicate and produce clones. The clones can then be examined for the desired gene sequence or function. The clones can also be used to generate a large quantity of protein by placing the gene into an expression vector that will cause it to be transcribed and translated into a product.
Scientists are using cloning to make new genetically engineered plants, animals, and microorganisms with desirable characteristics such as resistance to disease or the ability to produce useful biofuels. They are also experimenting with the possibility of using cloning to create stem cells that can grow into any type of cell or tissue, such as nerve cells to fix a spinal cord injury or insulin-making cells for diabetics.
What is DNA?
DNA is the genetic information inside our cells, forming all living things and giving them unique traits. It’s like the code for a video game or blueprints for a house, but it is also the material that determines what we will look like in the future and how our bodies will work.
DNA, which stands for deoxyribonucleic acid, is a long molecule that consists of two long strands wrapped around each other in a shape known as a double helix. Each strand has a sugar (deoxyribose) and phosphate molecule at the ends, with a sequence of four bases – adenine (A), cytosine (C), guanine (G), and thymine (T) – between them. The sequence of these bases, called the gene, is the information encoded in the DNA that tells how we will look and function.
Each strand of DNA has a matching complementary strand, which contains the same base sequence but in the opposite direction. The two strands are connected by chemical bonds between the bases, and each base pairs with its complement on the other strand – A with T and C with G. The pairing of complementary bases on the two strands is what gives DNA its characteristic “ladder” structure.
The strands of DNA are coiled tightly together to form structures called chromosomes, found inside each cell that makes up the human body. The chromosomes are arranged in the nucleus of each cell, where they control how the cell functions.
Scientists can make exact copies of DNA, or clones, in the laboratory. The cloned DNA can then be inserted into a carrier, such as bacteria or yeast, and each time the carrier reproduces, a new copy of the cloned gene will be made.
What is a Gene Sequence?
In molecular biology, a gene sequence is the DNA that encodes for a particular protein. A gene consists of an open reading frame (ORF) that starts with an initiation codon — usually, but not always, ATG — and ends with a termination codon, TAA, or TAG. The ORF is surrounded by intron and exon sequences.
To determine the gene sequence of a fragment of DNA, scientists use a technique called Sanger sequencing. In this method, the target DNA is copied many times, creating DNA fragments of different lengths. These fragments are then compared to each other, using overlapping segments, to assemble the complete gene sequence. Next-generation sequencing techniques are now used, which are much faster and less expensive than Sanger sequencing.
Molecular cloning is the process by which cells, tissues, or even whole organisms are identical to their original genetic material. Some clones already exist in nature. Single-celled organisms like bacteria make exact copies of themselves each time they reproduce, and humans produce identical twins each time a fertilized egg divides.
The most common applications of cloning involve entire animals, such as cattle or pigs. Scientists create clones of these animals to study diseases that occur in the species, and they can also be used to produce more milk or meat. Clones can even be used to “resurrect” a deceased pet, as was the case with CC, a cat that was cloned in 2001.
A newer application of cloning is reproductive cloning, which allows the creation of an entirely new animal from a sample of its original DNA. To do this, scientists first take a cell from the animal to be cloned and insert it into an egg that has had its own DNA removed. The egg then begins dividing as it would in a fertilized situation, and the resulting embryo can be transferred to a surrogate female who will give birth to the cloned animal.
What is a Vector?
Vectors are geometrical quantities that possess both magnitude and direction. An arrow with a tail and a head often represent them. Vectors are useful in math, physics, and engineering for representing spatial quantities such as displacements, forces, and velocity. Vectors are distinguished from scalar quantities with a magnitude but no direction, such as distance, time, or mass.
Vector magnitude is usually a real number with units of measurement, and vector direction is a direction angle. Vectors can be combined with each other to obtain a new vector with a different magnitude and direction. For example, the vector (or line) AB extends from point A to point B. The new vector is the sum of the vector AB with vector C, which starts at point A and ends at point B, plus the vector (line) BC, which is drawn from the head of vector A to the tail of vector B.
To qualify as a vector, a quantity must have both magnitude and direction and obey certain rules of combination. For example, vectors must be parallel or antiparallel to each other, and their cross-product must be zero. This is called the commutative law of vectors. Vectors must also be able to be added together in the same order: A + B = C.
In gene cloning, a fragment of DNA is used as a vector to insert the recombinant genetic sequence into a host cell. For this purpose, the fragment of DNA is split into smaller pieces with restriction enzymes. The resulting DNA fragments are inserted into a suitable cloning vector, such as the bacterial F plasmid or bacteriophage, which can transport the recombinant genetic sequence to a host bacterial cell.
What is a Host Cell?
As the name implies, a host cell is a cell that harbors a virus or other microorganism. Viruses are obligate intracellular parasites that depend on their host cells’ structural and synthetic machinery for survival. Host-virus interactions play a critical role in regulating both viral and cellular processes and are intimately connected to the fields of virology and cell biology.
Cloning is the production of exact copies (clones) of a gene, DNA sequence, or whole organism. The process of cloning is used in the lab to produce genes that can be tested or modified. It is also used in a number of ways to make living things, such as cells, animals, and plants, which do not normally exist.
During gene cloning, the DNA from an organism that contains the desired genetic sequence is split into gene-size fragments with restriction enzymes. These fragments are then inserted into self-replicating DNA elements called vectors, such as bacterial plasmids or bacteriophages, which transfer the recombinant DNA to host cells, where it can be copied many times over by normal cellular replication mechanisms.
This recombinant DNA is then used as a template to synthesize new DNA molecules containing the desired gene sequence. The synthesis of these new DNA molecules is carried out by a polymerase chain reaction, which is also known as PCR. The PCR procedure is an efficient way to generate large quantities of recombinant DNA in the lab.
Cloning is also used in the production of cells that can be turned into different types of cells, such as neurons to repair a damaged spinal cord or insulin-making cells to treat diabetes. It is even used to clone whole animals, such as mice and cattle. When done in a way that preserves the genetic integrity of the animal, the results are called embryonic clones, which can be fertilized and implanted into a surrogate female to develop into a live clone of the original animal.