MCAT Biochemistry Review

Chapter 6: DNA and Biotechnology

6.5 Recombinant DNA and Biotechnology

Now that we have reviewed the basics of DNA structure and function, we can discuss how this knowledge has been harnessed for a variety of research and treatment innovations. Recombinant DNA technology allows a DNA fragment from any source to be multiplied by either gene cloning or polymerase chain reaction (PCR). This provides a means of analyzing and altering genes and proteins. It also provides the reagents necessary for genetic testing, such as carrier detection (detecting heterozygote status for a particular disease) and prenatal diagnosis of genetic diseases; it is also useful for gene therapy. Additionally, this technology can provide a source of a specific protein, such as recombinant human insulin, in almost unlimited quantities. The process of creating recombinant DNA (by gene cloning) and its benefits are shown in Figure 6.18.

Figure 6.18. Cloning Recombinant DNA Cloning allows for production of recombinant proteins, or identification and characterization of DNA by increasing its volume and purity.

DNA CLONING AND RESTRICTION ENZYMES

DNA cloning is a technique that can produce large amounts of a desired sequence. Often, the DNA to be cloned is present in a small quantity and is part of a heterogeneous mixture containing other DNA sequences. The goal is to produce a large quantity of homogeneous DNA for other applications. Cloning requires that the investigator ligate the DNA of interest into a piece of nucleic acid referred to as a vector, forming a recombinant vector. Vectors are usually bacterial or viral plasmids that can be transferred to a host bacterium after insertion of the DNA of interest. The bacteria are then grown in colonies, and a colony containing the recombinant vector is isolated. This can be accomplished by ensuring that the recombinant vector also includes a gene for antibiotic resistance; antibiotics can then kill off all of the colonies that do not contain the recombinant vector. The resulting colony can then be grown in large quantities. Depending on the investigator's goal, the bacteria can then be made to express the gene of interest (generating large quantities of recombinant protein), or can be lysed to reisolate the replicated recombinant vectors (which can be processed by restriction enzymes to release the cloned DNA from the vector).

Restriction enzymes (restriction endonucleases) are enzymes that recognize specific double-stranded DNA sequences. These sequences are palindromic, meaning that the 5′ to 3′ sequence of one strand is identical to the 5′ to 3′ sequence of the other strand (in antiparallel orientation). Restriction enzymes are isolated from bacteria, which are their natural source. In bacteria, they act as part of a restriction and modification system that protects the bacteria from infection by DNA viruses. Once a specific sequence has been identified, the restriction enzyme can cut through the backbones of the double helix. Thousands of restriction enzymes have been studied and many are commercially available to laboratories, allowing us to process DNA in very specific ways. Some restriction enzymes produce offset cuts, yielding sticky ends on the fragments, as shown in Figure 6.19. Sticky ends are advantageous in facilitating the recombination of a restriction fragment with the vector DNA. The vector of choice can also be cut with the same restriction enzyme, allowing for the fragments to be inserted directly into the vector.

Figure 6.19. A Restriction Enzyme (EcoRI) Creating Sticky Ends Restriction enzymes cut at palindromic sequences, such as GAATTC.

DNA vectors contain at least one sequence, if not many, recognized by restriction enzymes. A vector also requires an origin of replication and at least one gene for antibiotic resistance to allow for selection of colonies with recombinant plasmids, as described above. The formation of a recombinant plasmid is shown in Figure 6.20.

Figure 6.20. Formation of a Recombinant Plasmid Vector ori: origin of replication; ampr: gene for resistance to ampicillin (an antibiotic).

DNA LIBRARIES AND cDNA

DNA cloning can be used to produce DNA libraries. DNA libraries are large collections of known DNA sequences; in sum, these sequences could equate to the genome of an organism. To make a DNA library, DNA fragments, often digested randomly, are cloned into vectors and can be utilized for further study. Libraries can consist of either genomic DNA or cDNA. Genomic libraries contain large fragments of DNA, and include both coding (exon) and noncoding (intron) regions of the genome. cDNA (complementary DNA) libraries are constructed by reverse-transcribing processed mRNA, as shown in Figure 6.21. As such, cDNA lacks noncoding regions, such as introns, and only includes the genes that are expressed in the tissue from which the mRNA was isolated. For that reason, these libraries are sometimes called expression libraries. While genomic libraries contain the entire genome of an organism, genes may by chance be split into multiple vectors. Therefore, only cDNA libraries can be used to reliably sequence specific genes and identify disease-causing mutations, produce recombinant proteins (such as insulin, clotting factors, or vaccines), or produce transgenic animals. Several of these applications are discussed in more detail in subsequent sections of this chapter. Table 6.3 contrasts some of the characteristics of genomic and cDNA libraries.

Figure 6.21. Cloning Expressed Genes by Producing cDNA

 

Genomic Libraries

cDNA (Expression) Library

Source of DNA

Chromosomal DNA

mRNA (cDNA)

Enzymes to make library

Restriction endonuclease DNA ligase

Reverse transcriptase DNA ligase

Contains nonexpressed sequences of chromosomes

Yes

No

Cloned genes are complete sequences

Not necessarily

Yes

Cloned genes contain introns

Yes

No

Promoter and enhancer sequences present

Yes, but not necessarily in same clone

No

Gene can be expressed in cloning host (recombinant proteins)

No

Yes

Can be used for gene therapy or constructing transgenic animals

No

Yes

Table 6.3. Comparison of Genomic and cDNA (Expression) Libraries

HYBRIDIZATION

Another tool often used by researchers is called hybridization. Hybridization is the joining of complementary base pair sequences. This can be DNA–DNA recognition or DNA–RNA recognition. This technique uses two single-stranded sequences and is a vital part of polymerase chain reaction and Southern blotting.

Polymerase Chain Reaction

Polymerase chain reaction (PCR) is an automated process that can produce millions of copies of a DNA sequence without amplifying the DNA in bacteria. PCR is used to identify criminal suspects, familial relationships, and disease-causing bacteria and viruses. Knowing the sequences that flank the desired region of DNA allows for the amplification of the sequence in between. A PCR reaction requires primers that are complementary to the DNA that flanks the region of interest, nucleotides (A, T, C, and G), and DNA polymerase. The reaction also needs heat to cause the DNA double helix to melt apart (denature). Unfortunately, the DNA polymerase found in the human body does not work at high temperatures. Thus, the DNA polymerase from Thermus aquaticus, a bacteria that thrives in the hot springs of Yellowstone National Park at 70°C, is used instead.

BRIDGE

PCR provides a great example of the temperature dependence of enzymes. While human DNA polymerase denatures at the high temperatures required in PCR, the DNA polymerase from Taquaticus functions optimally at these temperatures. Refer to Chapter 2 of MCAT Biochemistry Review for more on the link between temperature and enzyme activity.

Gel Electrophoresis and Southern Blotting

Gel electrophoresis is a technique used to separate macromolecules, such as DNA and proteins, by size and charge. Electrophoresis of proteins was discussed in detail in Chapter 3 of MCAT Biochemistry Review, but DNA can be separated in a similar way. All molecules of DNA are negatively charged because of the phosphate groups in the backbone of the molecule, so all DNA strands will migrate toward the anode of an electrochemical cell. The preferred gel for DNA electrophoresis is agarose gel, and—just like proteins in polyacrylamide gel—the longer the DNA strand, the slower it will migrate in the gel.

Gel electrophoresis is often used while performing a Southern blot. A Southern blot is used to detect the presence and quantity of various DNA strands in a sample. DNA is cut by restriction enzymes and then separated by gel electrophoresis. The DNA fragments are then carefully transferred to a membrane, retaining their separation. The membrane is then probed with many copies of a single-stranded DNA sequence. The probe will bind to its complementary sequence and form double-stranded DNA. Probes are labeled with radioisotopes or indicator proteins, both of which can be used to indicate the presence of a desired sequence.

DNA SEQUENCING

DNA sequencing has revolutionized the world that we live in. The applications of this technique are far-reaching, from the medical field to criminal courts. A basic sequencing reaction contains the main players from replication, including template DNA, primers, an appropriate DNA polymerase, and all four deoxyribronucleotide triphosphates. In addition, a modified base called a dideoxyribonucleotide is added in lower concentrations. Dideoxyribonucleotides (ddATP, ddCTP, ddGTP, and ddTTP) contain a hydrogen at C-3', rather than a hydroxyl group; thus, once one of these modified bases has been incorporated, the polymerase can no longer add to the chain. Eventually the sample will contain many fragments (as many as the number of nucleotides in the desired sequence), each one of which terminates with one of the modified bases. These fragments are then separated by size using gel electrophoresis. The last base for each fragment can be read, and because gel electrophoresis separates the strands by size, the bases can easily be read in order.

REAL WORLD

The Human Genome Project, initiated in 1991, involved the identification of all 3 billion base pairs of the human DNA sequence. The first draft of this project was completed in 2000. This project demonstrated that although humans appear to be quite different from each other, the sequence of our DNA is, in reality, highly conserved. On average, two unrelated individuals still share over 99.9% of their DNA sequences.

APPLICATIONS OF DNA TECHNOLOGY

In addition to its utility as a research tool, DNA biotechnology has led to a number of therapeutic breakthroughs. This is likely only the beginning, as biotechnology continues to be an active area of research.

Gene Therapy

Gene therapy now offers potential cures for individuals with inherited diseases. Gene therapy is intended for diseases in which a given gene is mutated or inactive, giving rise to pathology. By transferring a normal copy of the gene into the affected tissues, the pathology should be fixed, essentially curing the individual. For instance, about half of children with severe combined immunodeficiency (SCID) have a mutation in the gene encoding the γ chain common to several of the interleukin receptors. By placing a working copy of the gene for the γ chain into a virus, one can transmit the functional gene into human cells. The first successful case of gene therapy was for SCID (caused by a different mutation) in 1990.

For gene replacement therapy to be a realistic possibility, efficient gene delivery vectors must be used to transfer the cloned gene into the target cells' DNA. Because viruses naturally infect cells to insert their own genetic material, most gene delivery vectors in use are modified viruses. A portion of the viral genome is replaced with the cloned gene such that the virus can infect but not complete its replication cycle, as shown in Figure 6.22. Randomly integrated DNA poses a risk of integrating near and activating a host oncogene. Among the children treated for SCID, a small number have developed leukemias (cancers of the white blood cells).

Figure 6.22. Retroviral Gene Therapy The example given here uses a retrovirus, but other viruses may also be used for gene therapy.

Transgenic and Knockout Mice

Once DNA has been isolated, it can be introduced into eukaryotic cells. Transgenic mice are altered at their germ line by introducing a cloned gene into fertilized ova or into embryonic stem cells. The cloned gene that is introduced is referred to as a transgene. If the transgene is a disease-producing allele, the transgenic mice can be used to study the disease process from early embryonic development through adulthood. A similar approach can be used to produce knockout mice, in which a gene has been intentionally deleted (knocked out). These mice provide valuable models in which to study human diseases.

There are different approaches to developing transgenic mice. A cloned gene may be microinjected into the nucleus of a newly fertilized ovum. Rarely, the gene may subsequently incorporate into the nuclear DNA of the zygote. The ovum is implanted into a surrogate mother, and, if successful, the resulting offspring will contain the transgene in all of their cells, including their germ line cells (gametes). Consequently, the transgene will also be passed to their offspring. The transgene coexists in the animals with their own copies of the gene, which have not been deleted. This approach is useful for studying dominant gene effects but is less useful as a model for recessive disease because the number of copies of the gene that insert into the genome cannot be controlled; the transgenic mice may each contain a different number of copies of the transgene. This method is demonstrated in Figure 6.23.

Figure 6.23. Creation of a Transgenic Mouse

Embryonic stem cell lines can also be used for developing transgenic mice. Advantages of using stem cell lines are that the cloned genes can be introduced in cultures, and that one can select for cells with the transgene successfully inserted. The altered stem cells are injected into developing blastocysts and implanted into surrogate mothers. The blastocyst itself is thus composed of two types of stem cells: the ones containing the transgene and the original blastocyst cells that lack the transgene. The resulting offspring is a chimera, meaning that it has patches of cells, including germ cells, derived from each of the two lineages. This is evident if the two cell lineages (transgenic cells and host blastocyst) come from mice with different coat colors. The chimeras will have patchy coats of two colors, allowing them to be easily identified. These chimeras can then be bred to produce mice that are heterozygous for the transgene and mice that are homozygous for the transgene.

SAFETY AND ETHICS

The different procedures and techniques that have been reviewed provide great insight for researchers in many different fields of study. However, it is also important to acknowledge the potential risks associated with these technologies. Safety concerns such as increased resistance in viruses and bacteria can impact both humans and the environment in which we live. Ethical dilemmas arise: is it ethical to test for life-threatening genetic diseases and potentially terminate a pregnancy based on the results? What about testing for eye or hair color? What are the ethical questions around choosing human test subjects? If a disease-causing gene were found in one individual of a family, does this need to be communicated to other relatives at risk, potentially violating principles of privacy? Is it permissible to carry out potentially risky therapy in an individual whose illness makes him or her unable to communicate? The medical community and bioethicists at large continue to wrestle with this question: how much should we meddle with our own genetic makeup?

REAL WORLD

The story of Jesse Gelsinger is one particularly harrowing example of the problems that biotechnology can create. Diagnosed with mild ornithine transcarbamylase (OTC) deficiency, a defect of the urea cycle, Gelsinger volunteered for a clinical trial of gene therapy. During the trial (which was also fraught with questionable consent practices), Gelsinger died from a massive immune response to the viral vector, leading to multiple organ failure and brain death. He was 18 years old.

MCAT Concept Check 6.5:

Before you move on, assess your understanding of the material with these questions.

1.    When creating a DNA library, what are some of the advantages of genomic libraries? What about cDNA libraries?

·        Genomic:

·        cDNA:

2.    What does PCR accomplish for a researcher? What about Southern blotting?

·        PCR:

·        Southern blotting:

3.    During DNA sequencing, why does the DNA polymer stop growing once a dideoxyribonucleotide is added?

4.    What is the difference between a transgenic and a knockout mouse?