Gene Cloning
You probably have heard of cloning. Whereas cloning of humans has many ethical issues associated with it, the cloning of genes has been ongoing for well over 30 years, with cloning of animals occurring more recently. Gene cloning, also known as molecular cloning, refers to the process of isolating a DNA sequence of interest for the purpose of making multiple copies of it. The identical copies are clones. In 1973, Stanley Cohen and Herbert Boyer developed techniques to make recombinant DNA, a form of artificial DNA.
Recombinant DNA is engineered through the combination of two or more DNA strands, combining DNA sequences which would not normally occur together. In other words, selected DNA (or the DNA of "interest") is inserted into an existing organismal genome, such as a bacterial plasmid DNA or some other sort of vector. The recombinant DNA can then be inserted into another cell, such as a bacterial cell, for amplification and possibly production of the resulting protein. This process is called transformation, the genetic alteration of a cell resulting from the uptake, incorporation, and expression of foreign genetic material. Recombinant DNA technology was made possible by the discovery of restriction endonucleases.
Restriction Enzyme Digestion and Ligation
In the classical restriction enzyme digestion and ligation cloning protocols, cloning of any DNA fragment essentially involves four steps:
isolation of the DNA of interest (or target DNA)
ligation
transfection (or transformation)
a screening/selection procedure.
For an overview of cloning, see http://www.hhmi.org/biointeractive/media/DNAi_genetic_eng-sm.mov.
Isolation of DNA
Initially, the DNA fragment to be cloned needs to be isolated. This DNA of interest may be a gene, part of a gene, a promoter, or another segment of DNA, and is frequently isolated by the Polymerase Chain Reaction (PCR) or restriction enzyme digestion. A restriction enzyme (or restriction endonuclease) is an enzyme that cuts double-stranded DNA at a specific sequence (Table below). The enzyme makes two incisions, one through each strand of the double helix, without damaging the nitrogenous bases. This produces either overlapping ends (also known as sticky ends) or blunt ends.
A. G↓AATTC B. CCC↓GGG
CTTAA↑G GGG↑CCC
A. EcoRI digestion produces overlapping "sticky" ends: The enzyme cleaves between the G and A on both strands. B. SmaII restriction enzyme cleavage produces "blunt" ends. The enzyme cleaves between the G and C on both strands.
(Source: Created by: Doug Wilkin, License: CC-BY-SA)
The 1978 Nobel Prize in Medicine was awarded to Daniel Nathans and Hamilton Smith for the discovery of restriction endonucleases. The first practical use of their work was the manipulation of E. coli bacteria to produce human insulin for diabetics.
Ligation
Once the DNA of interest is isolated, a ligation procedure is necessary to insert the amplified fragment into a vector to produce the recombinant DNA molecule. Restriction fragments (or a fragment and a plasmid/vector) can be spliced together, provided their ends are complementary. Blunt end ligation is also possible.
The plasmid or vector (which is usually circular) is digested with restriction enzymes, opening up the vector to allow insertion of the target DNA. The two DNAs are then incubated with DNA ligase, an enzyme that can attach together strands of DNA with double strand breaks. This produces the recombinant DNA molecule. Figure below depicts a plasmid with two additional segments of DNA ligated into the plasmid, producing the recombinant DNA molecule. Figure below depicts DNA before and after ligation.
Figure 10.3
This image shows a line drawing of a plasmid. The plasmid is drawn as two concentric circles that are very close together, with two large segments and one small segment depicted. The two large segments (1 and 2) indicate antibiotic resistances usually used in a screening procedure, and the small segment (3) indicates an origin of replication. The resulting DNA is a recombinant DNA molecule.
Figure 10.4
Sticky ends produced by restriction enzyme digestion can be joined with the enzyme DNA ligase.
Transfection and Selection
Following ligation, the recombinant DNA is placed into a host cell, usually bacterial, in a process called transfection or transformation. Finally, the transfected cells are cultured. Many of these cultures may not contain a plasmid with the target DNA as the transfection process is not usually 100% successful, so the appropriate cultures with the DNA of interest must be selected. Many plasmids/vectors include selectable markers - usually some sort of antibiotic resistance (Figure above). When cultures are grown in the presence of an antibiotic, only bacteria transfected with the vector containing resistance to that antibiotic should grow. However, these selection procedures do not guarantee that the DNA of insert is present in the cells. Further analysis of the resulting colonies is required to confirm that cloning was successful. This may be accomplished by means of a process known as PCR (see below) or restriction fragment analysis, both of which need to be followed by gel electrophoresis and/or DNA sequencing (DNA sequence analysis).
DNA sequence analysis (the analysis of the order of the nitrogenous bases that make up the DNA), PCR, or restriction fragment analysis will all determine if the plasmid/vector contains the insert. Restriction fragment analysis is digestion of isolated plasmid/vector DNA with restriction enzymes. If the isolated DNA contains the target DNA, that fragment will be excised by the restriction enzyme digestion. Gel electrophoresis will separate DNA molecules based on size and charge. Examples are shown in Figure below.
Gel Electrophoresis
Gel electrophoresis is an analytical technique used to separate DNA fragments by size and charge. Notice in Figure below that the "gels" are rectangular in shape. The gels are made of a gelatin-like material of either agarose or polyacrylamide. An electric field, with a positive charge applied at one end of the gel, and a negative charge at the other end, forces the fragments to migrate through the gel. DNA molecules migrate from negative to positive charges due to the net negative charge of the phosphate groups in the DNA backbone. Longer molecules migrate more slowly through the gel matrix. After the separation is completed, DNA fragments of different lengths can be visualized using a fluorescent dye specific for DNA, such as ethidium bromide. The resulting stained gel shows bands correspond to DNA molecules of different lengths, which also correspond to different molecular weights. Band size is usually determined by comparison to DNA ladders containing DNA fragments of known length. Gel electrophoresis can also be used to separate RNA molecules and proteins.
Figure 10.5
Agarose gel following agarose gel electrophoresis on UV light box: In the gel with UV illumination (left), the ethidium bromide stained DNA glows pink; Right, photo of a gel. Far left: DNA ladder of fragments of known length. Lane 1: A PCR product of just over 500 bases. Lane 2: Restriction digest showing the 500 base fragment cut from a 4.5 kb plasmid vector.
The Polymerase Chain Reaction
The Polymerase Chain Reaction (PCR) is used to amplify specific regions of a DNA strand millions of times. A region may be a number of loci, a single gene, a part of a gene, or a non-coding sequence. This technique produces a useful quantity of DNA for analysis, be it medical, forensic or some other form of analysis. Amplification of DNA from as little as a single cell is possible. Whole genome amplification is also possible.
PCR utilizes a heat stable DNA polymerase, Taq polymerase, named after the thermophilic bacterium Thermus aquaticus, from which it was originally isolated. T. aquaticus is a bacterium that lives in hot springs and hydrothermal vents, and Taq polymerase is able to withstand the high temperatures required to denature DNA during PCR (discussed below). Taq polymerase’s optimum temperature for activity is between 75°C and 80°C. Recently other DNA polymerases have also been used for PCR.
A basic PCR involves a series of repeating cycles involving three main steps (see Figure below):
denaturation of the
double stranded DNA
annealing of specific oligonucleotide primers
extension of the primers to amplify the region of DNA of interest
These steps will be discussed in additional detail below.
The oligonucleotide primers are single stranded pieces of DNA that correspond to the 5’ and 3’ ends of the DNA region to be amplified. These primers will anneal to the corresponding segment of denatured DNA. Taq Polymerase, in the presence of free deoxynucleotide triphosphates (dNTPs), will extend the primers to create double stranded DNA. After many cycles of denaturation, annealing and extension, the region between the two primers will be amplified.
The PCR is commonly carried out in a thermal cycler, a machine that automatically allows heating and cooling of the reactions to control the temperature required at each reaction step (see below). The PCR usually consists of a series of about 30 to 35 cycles. Most commonly, PCR is carried out in three repeating steps, with some modifications for the first and last step.
PCR is usually performed in small tubes or wells in a tray, each often beginning with the complete genome of the species being studied. As only a specific sequence from that genome is of interest, the sequence specific primers are targeted to that sequence. PCR is done with all the building blocks necessary to create DNA: template DNA, primers, dNTPs, and a polymerase.
The three basic steps of PCR (Figure below) are:
Denaturation step: This step is the first regular cycling event and consists of heating the reaction to 94 - 98°C for 30 to 60 seconds. It disrupts the hydrogen bonds between complementary bases of the DNA strands, yielding single strands of DNA.
Annealing step: The reaction temperature is lowered to 50-65°C for 30 to 60 seconds, allowing annealing of the primers to the single-stranded DNA template. Stable hydrogen bonds form between the DNA strand (the template) and the primers when the primer sequence very closely matches the complementary template sequence. Primers are usually 17 - 22 nucleotides long and are carefully designed to bind to only one site in the genome. The polymerase binds to the primer-template hybrid and begins DNA synthesis.
Extension step: A temperature of around 72°C is used for this step, which is close to the optimum temperature of Taq polymerase. At this step the Taq polymerase extends the primer by adding dNTPs, using one DNA strand as a template to create a the other (new) DNA strand. The extension time depends on the length of the DNA fragment to be amplified. As a standard, at its optimum temperature, the DNA polymerase will polymerize a thousand bases in one minute.
Figure 10.6
PCR: A repeating cycle of denaturation (1), annealing (2), and extension (3). Notice that initially there is a double strand of DNA, and after denaturation, the DNA is single stranded. In the annealing step (2), single stranded primers bind. These primers are extended by Taq Polymerase, represented by the green ball (3).
Utilizing the PCR, DNA can be amplified millions of times to generate quantities of DNA that can be used for a number of purposes. These include the use of DNA for prenatal or genetic testing, such as testing for a specific mutation. PCR has revolutionized the fields of biotechnology, human genetics, and a number of other sciences. Many of the applications will be discussed in the following lesson. PCR was developed in 1983 by Kary Mullis. Due to the importance of this process and the significance it has had on scientific research, Dr. Mullis was awarded the Nobel Prize in Chemistry in 1993, just 10 years after his discovery.
To say that PCR, molecular cloning and the Human Genome Project has revolutionized biology and medicine would be an understatement. These efforts have led to numerous accolades, including Nobel prizes, and more may follow. Some of the ways that these discoveries have shaped our lives are the focus of the next lesson.
Lesson Summary
Biotechnology is technology based on biological applications, combining many features of Biology including genetics, molecular biology, biochemistry, embryology, and cell biology.
The goal of the Human Genome Project is to understand the genetic make-up of the human species by determining the DNA sequence of the human genome and the genome of a few model organisms.
Gene cloning, also known as molecular cloning, refers to the process of isolating a DNA sequence of interest for the purpose of making multiple copies of it (cloning).
Classic gene cloning involves the following steps:
Restriction enzyme digestion and ligation
Isolation of DNA
Ligation
Transfection and Selection
Gel electrophoresis
The Polymerase Chain Reaction (PCR) is used to amplify millions of times specific regions of a DNA strand. This can be a number of loci, a single gene, a part of a gene, or a non-coding sequence.
PCR usually involves the following steps:
Denaturation step
Annealing step
Extension step
Review Questions
Why are biotechnology and DNA technology considered the same?
What are the goals of the Human Genome Project?
Is the DNA sequence information generated by the HGP available to anyone, and if so, how?
How are gene cloning and recombinant DNA related?
Describe the process of gene cloning.
How does gel electrophoresis analyze DNA?
What is PCR?
What allows PCR to be done at high temperatures?
Describe the PCR process.
Further Reading / Supplemental Links
http://www.genome.gov/HGP
http://www.ornl.gov/sci/techresources/Human_Genome/home.shtml
http://biotech.about.com/od/cloning/tp/DNAcloning.htm
http://croptechnology.unl.edu/viewLesson.cgi?LessonID=957884601
http://www.pcrstation.com/
http://nobelprize.org/educational_games/chemistry/pcr/index.html
http://en.wikipedia.org
http://www.vtaide.com/png/cloning.htm
Vocabulary
biotechnology
Technology based on biological applications.
gel electrophoresis
An analytical technique used to separate DNA fragments by size and charge.
Genbank
The U.S. National Center for Biotechnology Information, part of the National Institutes of Health, which maintains the genomic sequences in a database.
gene cloning
The process of isolating a DNA sequence of interest for the purpose of making multiple copies of it.
The Human Genome Project
A project to understand the genetic make-up of the human species by determining the DNA sequence of the human genome and the genome of a few model organisms.
plasmid (or vector)
A small circular piece of DNA that carries the recombinant DNA into a host organism for cloning.
polymerase chain reaction (PCR)
A repeating series of cycles used to amplify millions of times specific regions of a DNA strand.
recombinant DNA
Engineered through the combination of two or more DNA strands that combine DNA sequences which would not normally occur together.
restriction enzyme (or restriction endonuclease)
An enzyme that cuts double-stranded DNA.
Taq polymerase
Named after the thermophilic bacterium Thermus aquaticus from which it was originally isolated, is the heat-stable polymerase used in the PCR reaction.
Points to Consider
The Human Genome Project, gene cloning, and PCR are some of the most remarkable scientific achievements of the recent past. But how can these milestones make our lives better?
Medicine and food science are just two of the categories that benefit from biotechnology. Speculate on how our lives are made better by these achievements.
Lesson 10.2: Biotechnology
Lesson Objectives
Describe various applications of biotechnology as related to medicine, agriculture and forensic science.
; How is DNA technology related to genetic testing and prenatal diagnosis?
Why is biotechnology so important in agriculture?
Why is DNA analysis the most important tool of the forensic scientist?
Describe forensic STR analysis.
Discuss some of the ELSI associated with biotechnology.
Introduction
Scientists have sequenced a consensus version of the human genome. Now what? Do we know what all the genes are or what they do? Not yet. Do we know what phenotypes are associated with mutations in the genes? For many genes, or even most genes, we do not. Do we even know exactly how many genes we have? Not exactly. And we are far away from knowing what makes us all unique. So how does this information help us? The Human Genome Project has been labeled a landmark scientific event. But what can we do with this information?
CK-12 Biology I - Honors Page 42