Molecular Genetics 1: DNA Structure and Replication

The three letters “DNA” have now become associated with crime solving, paternity testing, human identification, and checking for genetic abnormalities. DNA can be retrieved from many biological samples, such as hair, blood, or saliva. With the exception of identical twins, each person’s DNA is unique, making it is possible to detect differences between human beings on the basis of their unique DNA sequence.

DNA analysis has many practical applications beyond forensics and paternity testing, such as tracing genealogy and identifying pathogens. In the medical field, DNA is used in diagnostics, new vaccine development, and cancer therapy. It is now possible to determine predisposition to many diseases by analysing genes.

DNA is the genetic material passed from parent to offspring for all life on Earth. The technology of molecular genetics developed in the last half century has enabled us to see deep into the history of life to deduce the relationships between living things in ways never thought possible. It also allows us to understand the workings of evolution in populations of organisms. Over a thousand species have had their entire genome sequenced, and there have been thousands of individual human genome sequences completed. 

Decoding these sequences will allow scientists insight into the origins and mechanisms of human diseases and provide a genetic map of evolutionary relationships with other life forms on the Tree of Life. 

Finally, molecular genetic techniques have revolutionised plant and animal breeding for human agricultural needs. Modern advances in biotechnology are built upon decades of foundational research, beginning with the groundbreaking discovery of DNA’s double helix structure by James Watson and Francis Crick in 1953. Subsequent research have unravelled the intricate mechanisms of DNA replication and the complex process leading to translation of genetic information into functional proteins within cells.

Introduction

The genetic (hereditary) material for all living things is composed of deoxyribonucleic acid, commonly known as DNA. The structure of DNA must enable this substance to store coded information that control the biological function of cells. Genetic material transmits this hereditary information in a stable form through the highly accurate and controlled replication of DNA. While DNA is capable of change (a topic we’ll explore further when discussing mutations), the replication process ensures that genetic information is faithfully copied, so all progeny cells receive the same instructions. And there are a lot of instructions! Remarkably, if you laid out the total length of DNA in all human chromosomes it would exceed 2 meters in length! To fit within the tiny space of the cell nucleus, this DNA is tightly packaged into structures called chromosomes.

History of Chromatin

The speculation that chromosomes might be the key to understanding heredity led several scientists to examine Mendel’s publications and reevaluate his model in terms of chromosome behaviour during mitosis and meiosis. In 1902, Theodor Boveri observed that proper sea urchin embryonic development does not occur unless chromosomes are present. That same year, Walter Sutton observed chromosome separation into daughter cells during meiosis (Fig 7.1). Together, these observations led to the Chromosomal Theory of Inheritance, which identified chromosomes as the genetic material responsible for Mendelian inheritance.

History of DNA Structure 

DNA Structure

DNA is a polymer made of nucleotide sub-units.

The important components of the nucleotide are  a nitrogenous (nitrogen-bearing) base, a 5-carbon sugar (pentose), and a phosphate group (Figure 7.3).

The nucleotide is named depending on the nitrogenous base. In DNA, nitrogenous base can be a purine such as adenine (A) and guanine (G), or a pyrimidine such as cytosine (C) and thymine (T). The pentose sugar molecule in DNA is known as deoxyribose, named for the removal of a hydroxy group “deoxy”- from a “ribose” molecule, another monosaccharide molecule. Ribose is the sugar molecule which makes up RNA (which we will discuss later) and the “energy molecule” adenosine triphosphate (ATP).  

Visual Connection 

The images above (Fig 7.4) illustrate the five bases of DNA and RNA. Examine the images and explain why these are called “nitrogenous bases.” How are the purines different from the pyrimidines? How is one purine or pyrimidine different from another, e.g., adenine from guanine? How is a nucleoside different from a nucleotide?

The purines have a double ring structure with a six-membered ring fused to a five-membered ring. Pyrimidines are smaller in size; they have a single six-membered ring structure.

The sugar is deoxyribose in DNA and ribose in RNA. The carbon atoms of the five-carbon sugar are numbered 1′, 2′, 3′, 4′, and 5′ (1′ is read as “one prime”). The phosphate, which makes DNA and RNA acidic, is connected to the 5′ carbon of the sugar by the formation of an ester linkage between phosphoric acid and the 5′-OH group (an ester is an acid + an alcohol). In DNA nucleotides, the 3′ carbon of the sugar deoxyribose is attached to a hydroxyl (OH) group. In RNA nucleotides, the 2′ carbon of the sugar ribose also contains a hydroxyl group. The base is attached to the 1’carbon of the sugar.

The nucleotides combine with each other to produce phosphodiester bonds. The phosphate residue attached to the 5′ carbon of the sugar of one nucleotide forms a second ester linkage with the hydroxyl group of the 3′ carbon of the sugar of the next nucleotide, thereby forming a 5′-3′ phosphodiester bond. In a polynucleotide, one end of the chain has a free 5′ phosphate, and the other end has a free 3′-OH. These are called the 5′ and 3′ ends of the chain.

A base attached to a five-carbon sugar represents a nucleoside. A nucleotide is composed of one, two, or three phosphate groups attached to a nucleoside.

Base pairing takes place between a purine and pyrimidine on opposite strands, so that A pairs with T, and G pairs with C (suggested by Chargaff’s Rules). Thus, adenine and thymine are complementary base pairs, and cytosine and guanine are also complementary base pairs. The base pairs are stabilised by hydrogen bonds: adenine and thymine form two hydrogen bonds and cytosine and guanine form three hydrogen bonds. The two strands are anti-parallel in nature; that is, the 3′ end of one strand faces the 5′ end of the other strand. The sugar and phosphate of the nucleotides form the backbone of the structure, whereas the nitrogenous bases are stacked inside, like the rungs of a ladder.

Each base pair is separated from the next base pair by a distance of 0.34 nm, and each turn of the helix measures 3.4 nm. Therefore, 10 base pairs are present per turn of the helix. The diameter of the DNA double-helix is 2 nm, and it is uniform throughout. Only the pairing between a purine and pyrimidine and the antiparallel orientation of the two DNA strands can explain the uniform diameter. The twisting of the two strands around each other results in the formation of uniformly spaced major and minor grooves (Figure 7.5).

 

Step 1: Initiating replication at the Ori

Helicase enzymes bind to the ori sequences and start to unwind the double stranded DNA. This establishes two replication forks (one shown in Figure 7.8a) and a helicase enzyme will be working to unwind the DNA at each fork as the forks move away from each other. The unwinding exposes single strands of DNA that are the template for building a new strand. Topoisomerase can be seen working to relieve tension ahead of the replication fork in Figure 7.8a.

Step 2: Priming DNA with RNA

The process of priming with primase and then replicating with DNA pol III happens at both replication forks (Figure 7.8a and b). DNA pol III enzymes can replicate new strands as fast as the DNA is unwound at each replication fork. The unwinding creates two template strands at each fork so primase enzyme must work to prime both old strands.

Step 3: Synthesis of leading and lagging strands

The double strands of DNA are anti-parallel and ‘run’ in opposite directions and DNA pol III can only work in one direction (Figure 7.8c). Thus, there is continuous and discontinuous replication happening at each replication fork. The strand that can be replicated as helicase unwinds is the leading strand. The DNA pol III must replicate the other strand in the opposite direction so this strand will lag behind. (Figure 7.8c).

Both replication forks have a leading and a lagging strand of new DNA. The DNA nucleotide stretches between the red RNA primers would be the Okazaki fragments.

One of the first scientists to provide evidence to support this model of DNA replication was the team of Reiji and Tsuneko Okazaki in the 1960s. They used radioactive nucleotides to track the newly replicating fragments and found that many of the fragments were short. This fits with the discontinuous model.

As the four enzymes just described attend to their specific roles in replication, two double stranded DNA molecules with an old strand and a new strand are built. If the DNA pol III makes a mistake and adds the wrong, non-complementing nucleotide, the enzyme can proof read its work and replace these replication mistakes. When replication is perfect, the two double stranded DNA molecules will have identical sequences.

Step 4: Ligation of Okazaki fragments

The fifth enzyme that has a final role in completing replication is DNA ligase (not shown in Figure 7.8d and e).  This enzyme will seal the sugar phosphate bond between the last replacement nucleotide added by DNA pol I and the first nucleotide that had been added after priming by DNA pol III.  With this bond sealing work, the replication process is complete.

The two double stranded DNA molecules that get made will have identical sequences unless rare mistakes happen as the enzymes conduct their work.

Because eukaryotic chromosomes are linear, DNA replication comes to the end of a line in eukaryotic chromosomes. As you have learned, the DNA polymerase enzyme can add nucleotides in only one direction. In the leading strand, synthesis continues until the end of the chromosome is reached; however, on the lagging strand there is no place for a primer to be made for the DNA fragment to be copied at the end of the chromosome.

This presents a problem for the cell because the ends remain unpaired, and over time these ends get progressively shorter as cells continue to divide. The ends of the linear chromosomes are known as telomeres, which have repetitive sequences that do not code for a particular gene. As a consequence, it is telomeres that are shortened with each round of DNA replication instead of genes. For example, in humans, a six base-pair sequence, TTAGGG, is repeated 100 to 1000 times. The discovery of the enzyme telomerase (Figure 7.9) helped in the understanding of how chromosome ends are maintained. The telomerase attaches to the end of the chromosome, and complementary bases to the RNA template are added on the end of the DNA strand.

Once the lagging strand template is sufficiently elongated, DNA polymerase can now add nucleotides that are complementary to the ends of the chromosomes. Thus, the ends of the chromosomes are replicated.

Figure 7.9. The ends of linear chromosomes are maintained by the action of the telomerase enzyme. Source: OpenStax, Biology 2e, by Mary Ann Clark, Matthew Douglas, and Jung Choi, Image created by Samantha Fletcher, and Asha Rao, Texas A&M Biology Department,  used under a Creative Commons Attribution-NonCommerical–ShareAlike 4.0 International License.Telomerase is typically found to be active in germ cells, adult stem cells, and some cancer cells. For her discovery of telomerase and its action, Elizabeth Blackburn (Figure 7.10) received the Nobel Prize for Medicine and Physiology in 2009. Later research using HeLa cells (obtained from Henrietta Lacks) confirmed that telomerase is present in human cells. And in 2001, researchers including Diane L. Wright found that telomerase is necessary for cells in human embryos to rapidly proliferate.

DNA polymerase can make mistakes while adding nucleotides. It edits the DNA by proofreading every newly added base. Incorrect bases are removed and replaced by the correct base, and then polymerisation continues (Figure 7.11a). Most mistakes are corrected during replication, although when this does not happen, the mismatch repair mechanism is employed.

Mismatch repair enzymes recognise the wrongly incorporated base and excise it from the DNA, replacing it with the correct base (Figure 7.11b). In yet another type of repair, nucleotide excision repair, the DNA double strand is unwound and separated, the incorrect bases are removed along with a few bases on the 5′ and 3′ end, and these are replaced by copying the template with the help of DNA polymerase (Figure 7.11c). Nucleotide excision repair is particularly important in correcting thymine dimers, which are primarily caused by ultraviolet light. In a thymine dimer, two thymine nucleotides adjacent to each other on one strand are covalently bonded to each other rather than their complementary bases. If the dimer is not removed and repaired, it will lead to a mutation. Individuals with flaws in their nucleotide excision repair genes show extreme sensitivity to sunlight and develop skin cancers early in life.

 

Most mistakes are corrected; if they are not, they may result in a mutation—defined as a permanent change in the DNA sequence. Mutations in repair genes may lead to serious consequences like cancer.

Another type of repair mechanism, nucleotide excision repair, is similar to mismatch repair, except that it is used to remove damaged bases rather than mismatched ones. The repair enzymes replace abnormal bases by making a cut on both the 3′ and 5′ ends of the damaged base (Figure 7.12). The segment of DNA is removed and replaced with the correctly paired nucleotides by the action of DNA pol. Once the bases are filled in, the remaining gap is sealed with a phosphodiester linkage catalysed by DNA ligase. This repair mechanism is often employed when UV exposure causes the formation of pyrimidine dimers.

 

Polymerase Chain Reaction (PCR)

The polymerase chain reaction laboratory technique is used in a variety of applications to make copies of a specific DNA sequence. In 1983, Kary Mullis was driving along a Californian mountain road late one night. As a molecular biologist, Dr. Mullis was imagining a better way to study DNA.  This late-night thinking led to a revolutionary way to make laboratory copies of DNA molecules (Saiki et al. 1985, Mullis 1990).  In the decades since, the polymerase chain reaction or PCR, has become the standard method used for detecting specific DNA or RNA sequences.  Selling the equipment and reagent kits for PCR is a multi-billion-dollar business because DNA and RNA detection is critical information in many applications.

PCR is an In Vitro process; a series of chemical reactions that happen outside of a living cell.  This laboratory technique is modelled after an In vivo process, the living cell’s natural ability to replicate DNA during normal cell. Every living cell makes a duplicate copy of each chromosome before the cell is ready to divide. Figure 7.15 below illustrates the key parts of In vivo DNA replication that are the basis for PCR success.

There are other enzymes that play an important role in in vivo replication.  However, PCR works as an in vitro  DNA replication process by using just one of these enzymes.  Mullis imagined a chemical reagent and a temperature change step in the method that could perform the work of the other two enzymes.  It should be noted that because Dr. Kerry Mullis had learned about the details of in vivo DNA replication, he could create this science changing in vitro  method.

The name ‘Polymerase Chain Reaction’ represents the nature of the process. ‘Polymerase’ because DNA Polymerase III is required for constructing new DNA strands, just like in a living cell. ‘Chain Reaction’ describes repeating cycles of replication which target a specific segment of a chromosome and use a “copy the copies” progression each cycle that doubles the amount of DNA copies of a specific segment of DNA present each cycle. In just 20 cycles of the chain reaction, over one million (220) copies of that specific segment of DNA can be produced.  This is enough DNA to see with your naked eye.  The goal of PCR is to make millions of copies of a specific segment of DNA that all originate from a single DNA sample.

The five chemical components that must be added to a test tube for the PCR reaction to work, include a DNA template, DNA polymerase III enzyme, single stranded DNA primers, nucleotides, and reaction buffer.

1. The DNA template is a sample of DNA that contains the target sequence of DNA for copying. 
2. DNA pol III. There are two requirements for a suitable DNA polymerase enzyme for PCR. First, the enzyme must have a good activity rate around 75°C. Second, the enzyme should be able to withstand temperatures of 95-100°C without denaturing and losing activity.
3. Two primers. Primers are short oligonucleotides of DNA, usually around 20 base pairs in length.  Because the purpose of PCR is to amplify a specific section of DNA in the genome, such as a known gene, then primers of specific sequences must be used.  The geneticist planning the PCR reaction will design a forward primer to bind to one strand and a reverse primer that complements and binds to the other strand. The primer design process to select forward and reverse primers is requiring appropriate genetics thinking and is describe later in this reading. 
4. The four different deoxyribonucleotide triphosphates (dNTPs).  Adenine (A), guanine (G), cytosine (C), and thymine (T) are needed to provide the building blocks for DNA replication. DNA polymerase will add each complementary base to the new growing DNA strand according to the original strand’s sequence following normal A-T and C-G pairings.
5. Finally, a reaction buffer.  This creates a stable pH and provides the Mg2+ cofactor needed for DNA pol III activity. 

Three Temperature Cycles

A key insight to the success of PCR as an in vitro DNA replication process which generates millions of specific sequence copies was a three-temperature cycle which accomplish three parts of DNA replication: denaturation of the double stranded template, annealing of the primers to the single strands and extension of new strand synthesis by DNA pol III.

In general, a single PCR run will undergo 25-35 cycles. The first step for a single cycle is the denaturation step, in which the double-stranded DNA template molecule (Figure 7.16) is made single-stranded (Figure 7.17). The temperature for this step is typically in the range of 95-100°C, near boiling. The high heat breaks the hydrogen bonds between the strands but does not break the sugar-phosphate bonds that hold the nucleotides of a single strand together (Figure 7.17).  

The geneticist planning the PCR analysis must “design” the forward and reverse primers and then buy them from a vendor who can synthesise single stranded DNA that has a specific sequence and length. The two most important criteria for primer design are the following.

1. One primer must have a sequence that complements one of the template strands and the other primer must be complementary to the other strand. BOTH strands need to be primed for the replication process.
2. The primers must bind so that their 3′ ends are ‘pointing’ in the direction of the other primer. This ensures that the sequence between the primers is replicated in the PCR cycles. 

Extension: The final PCR step is when the DNA polymerase enzyme reads the template and connects new nucleotides to the primer’s 3’ end, extending a new complementary strand of DNA (Figure 7.19). Completion of the final step and the first cycle of PCR, will make two double stranded DNA copies from the original template DNA, doubling of the amount of DNA present.

The test tube is heated to around 75°C, optimising DNA pol. III activity and the newly synthesising DNA strand is extended as the template strand is read by DNA pol. III. The Extension step will run for a few minutes and this step completes one PCR cycle.

When PCR was first invented, scientists used water baths set at different temperatures and the hand transfer of test tubes at timed intervals to run the PCR reaction.  Once the technique became a proven technology for DNA analysis, engineers went to work to create PCR machines.  The instrument which heats and cools the DNA samples is called a thermal cycler (Figure 7.21). Each small tube or sample well in a plate contains all the chemical components needed for a PCR reaction.  Adding a specific sample to the reaction mix provides the template DNA. A thermal cycler can be programmed for specific temperatures and the amount of time spent at each temperature.  The engineered design of thermal cyclers to maximise the accurate replication of the targeted DNA in a small sample volume with the minimum amount of time can be critical in many applications of PCR.

Taq DNA polymerase

When Dr. Kerry Mullis ran the first PCR experiments, he needed to add a new sample of DNA pol III after each denaturation step. This was because the high temperature needed to denature the double stranded DNA template also denatured the DNA pol III protein structure.  The DNA pol III enzyme commonly available to molecular geneticists was from E. coli bacteria and this enzyme had no stability at near boiling temperatures.

Fortunately, biologists had been investigating Thermus aquaticus, (Taq) a thermophilic eubacterium found in hot springs (Chien et al. 1976). The Taq version of DNA pol III does not easily denature in the hot temperatures required in PCR; plus, it has a good efficiency, able to add 60 base pairs/sec at 70°C. Like all other DNA polymerases, Taq DNA pol III cannot begin DNA replication without the addition of a starting primer. Thus, the discovery of Taq DNA pol III and the commercial availability of this enzyme made PCR a more reliable and doable technology which hastened its application to science investigation and diagnostic testing.

Advantages of PCR

PCR quickly became the method of choice for many types of DNA analysis because of several advantages over other DNA detection methods.  The first; it is a simple procedure to set up and run. Fewer steps save time in getting a DNA analysis result.  The second is the sensitivity. A very small amount of template DNA in the sample can be detected.  Even just a few skin cells from one human hair contain enough DNA, making PCR useful in forensics.  The third is that it can be designed to accurately differentiate genetic samples that are different by as little as single nucleotide in the targeted sequence.  Finally, in application where large numbers of samples need to be subjected to the same PCR analysis, automation and robotic assistance allow the processing of many samples in a very short time. For example, an automated PCR would be critical for testing large numbers of people in hours during a pandemic.

Limitations of PCR

There are some drawbacks of using PCR that one should be aware of as well. First, the sequence of the gene or chromosome region being targeted is required.  This limitation is rapidly diminishing as gene and genome sequencing technology and sharing of this sequence through Internet data bases has emerged as the norm in genetic analysis.  Because of the size of the genomes of living things and a high conservation of gene sequences in many organisms, primers designed for a PCR test must be empirically tested with the proper controls. Biologists can easily generate false positive DNA from PCR that is caused by contamination or lack of specificity in primer design.

There may also be a need to optimise concentrations of each chemical component. For example, changing the amount of DNA template, MgCl2 and Taq polymerase can affect both the quantity and quality of bands produced. Some studies have shown that even the brand of Taq polymerase can affect results (Holden et al. 2003). Likewise, the temperature cycles may need to be fine-tuned for a specific PCR test. Finally, as an in vitro DNA replication method, PCR cannot replicate entire chromosomes.

Gel Electrophoresis

Because nucleic acids are negatively charged ions at neutral or alkaline pH in an aqueous environment, they can be moved by an electric field. Gel electrophoresis is a technique used to separate charged molecules on the basis of size and charge. The nucleic acids can be separated as whole chromosomes or as fragments. The nucleic acids are loaded into a slot at one end of a gel matrix, an electric current is applied, and negatively charged molecules are pulled toward the opposite end of the gel (the end with the positive electrode). Smaller molecules move through the pores in the gel faster than larger molecules; this difference in the rate of migration separates the fragments on the basis of size. The nucleic acids in a gel matrix are invisible until they are stained with a compound that allows them to be seen, such as a dye. Distinct fragments of nucleic acids appear as bands at specific distances from the top of the gel (the negative electrode end) that are based on their size (Figure 7.22). A mixture of many fragments of varying sizes appear as a long smear, whereas uncut genomic DNA is usually too large to run through the gel and forms a single large band at the top of the gel.

Restriction Enzymes/RFLP for genotype analysis

Restriction enzymes (also called restriction endonucleases) recognise specific DNA sequences and cut them in a predictable manner; they are naturally produced by bacteria as a defence mechanism against foreign DNA. Many restriction enzymes make staggered cuts in the two strands of DNA, such that the cut ends have a 2- to 4-nucleotide single-stranded overhang. The sequence that is recognised by the restriction enzyme is a four- to eight-nucleotide sequence that is a palindrome. Like with a word palindrome, this means the sequence reads the same forward and backward. In most cases, the sequence reads the same forward on one strand and backward on the complementary strand. When a staggered cut is made in a sequence like this, the overhangs are complementary (Figure 7.24).

Plasmids with foreign DNA inserted into them are called recombinant DNA molecules because they contain new combinations of genetic material. Proteins that are produced from recombinant DNA molecules are called recombinant proteins. Not all recombinant plasmids are capable of expressing genes. Plasmids may also be engineered to express proteins only when stimulated by certain environmental factors, so that scientists can control the expression of the recombinant proteins.

Some genetic markers that scientists use in generating genetic maps are restriction fragment length polymorphisms (RFLP), variable number of tandem repeats (VNTRs), microsatellite polymorphisms, and the single nucleotide polymorphisms (SNPs). We can detect RFLPs (sometimes pronounced “rif-lips”) when the DNA of an individual is cut with a restriction endonuclease that recognises specific sequences in the DNA to generate a series of DNA fragments, which we can then analyse using gel electrophoresis. Every individual’s DNA will give rise to a unique pattern of bands when cut with a particular set of restriction endonucleases. Scientists sometimes refer to this as an individual’s DNA “fingerprint.” Certain chromosome regions that are subject to polymorphism will lead to generating the unique banding pattern. VNTRs are repeated sets of nucleotides present in DNA’s non-coding regions.

Non-coding, or “junk,” DNA has no known biological function; however, research shows that much of this DNA is actually transcribed. While its function is uncertain, it is certainly active, and it may be involved in regulating coding genes. The number of repeats may vary in a population’s individual organisms. Microsatellite polymorphisms are similar to VNTRs, but the repeat unit is very small. SNPs are variations in a single nucleotide.

Because genetic maps rely completely on the natural process of recombination, natural increases or decreases in the recombination level of a given genome area affects mapping. Some parts of the genome are recombination hotspots; whereas, others do not show a propensity for recombination. For this reason, it is important to look at mapping information developed by multiple methods.

Real World Applications

Career Connection

Clinical Trial Coordinator

A clinical trial coordinator is the person managing the proceedings of the clinical trial. This job includes coordinating patient schedules and appointments, maintaining detailed notes, building the database to track patients (especially for long-term follow-up studies), ensuring proper documentation has been acquired and accepted, and working with the nurses and doctors to facilitate the trial and publication of the results. A clinical trial coordinator may have a science background, like a nursing degree, or other certification. People who have worked in science labs or in clinical offices are also qualified to become a clinical trial coordinator. These jobs are generally in hospitals; however, some clinics and doctor’s offices also conduct clinical trials and may hire a coordinator.

References

Chien, A., Edgar, D. B., & Trela, J. M. (1976). Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus. Journal of bacteriology, 127(3), 1550-1557. https://doi.org/10.1128/jb.127.3.1550-1557.1976

Holden, M.; Blasic, J.; Bussjaeger, L.; Kao, C.; Shokere, L.; Kendall, D.; Freese, L.; Jenkins, G. (2003). Evaluation of Extraction Methodologies for Corn Kernel (Zea mays) DNA for Detection of Trace Amounts of Biotechnology-Derived DNA. J. Agric. Food Chem. 51 (9): 2468–2474. https://doi.org/10.1021/jf0211130