biolodgy d1.1

Whenever cell division occurs, whether it is for growth or for repair of tissues through cell replacement, the new cells each need a copy of the organism’s DNA. Growth involves the addition of new cells to make the organism larger. Each new cell requires a complete copy of the organism’s DNA, which it acquires through DNA replication that must occur prior to cell division. In this way, an organism can grow from a single fertilised egg cell to a large multicellular organism with trillions of cells, most with an exact copy of the DNA from that original fertilised egg cell.

When repairing damaged tissues by replacing cells, the same processes of cell division and DNA replication occur. New cells need to be produced to replace those that were damaged or destroyed. We have all had a cut or scrape. As those wounds heal, new cells are produced to fill in the gap created when the injury occurred. Each of these cells will be an exact genetic copy of those around them. They will also be differentiated to the same cell type by the gene expression within those cells.

The DNA of an organism contains the instructions for that organism. The base sequences contained within the DNA need to be copied exactly for the new cells to function properly. The process of DNA replication is able to copy billions of base pairs with amazing accuracy and at incredible speeds. The DNA in one of your cells can be copied completely in about an hour.The replication of DNA is semi-conservative. As one double strand of DNA is replicated, each new double strand of DNA that is produced contains one strand of the original DNA and one strand of newly synthesised DNA (Figure 1). This happens because each strand of the original DNA molecule acts as a template for the new strand to be built from. The complementary base pairing rule of DNA then ensures that the new strands that are built are exact copies of the original. The complementary base pairing rule states that the base adenine (A) always binds with the base thymine (T), and the base cytosine (C) always binds with the base guanine (G).

DNA replication is an essential process, and it must be carried out with tremendous accuracy to ensure that the new DNA functions exactly as the original. Enzymes play a key role in this process.

The process can be divided into separate steps. Eukaryotic DNA is normally supercoiled by being tightly wound around histones to form nucleosomes. This helps package the DNA and allows it to fit better within the nucleus. Prokaryotic DNA is not associated with these proteins and is referred to as ‘naked’ DNA. In eukaryotes like us, the first step in DNA replication is to unwind the coils to make the strands accessible to enzymes. The enzyme helicase then unwinds the double helix and separates the two DNA strands by breaking the hydrogen bonds between the bases. This separation of the strands exposes the bases usually protected within the molecule.

Once the strands are separated and the bases exposed, another enzyme called DNA polymerase can start its job. DNA polymerase will move along the separate DNA strands, using them as templates. It will then begin building a new strand of DNA by placing and attaching free nucleotides in a chain.

PCR stands for polymerase chain reaction. It is a technique used to amplify small fragments of DNA. The discovery of this technique has revolutionised medical science, forensic science and molecular biology. PCR has enabled scientists to clone genes, to work with minute amounts of DNA found at crime scenes, to identify the dead and, perhaps most extraordinarily, to sequence the DNA of extinct species and other life forms.

The desired section of DNA is placed in a reaction chamber that contains:

many free nucleoside triphosphates

primers that will allow replication to occur from the desired point, and

a special heat-stable version of DNA polymerase called Taq polymerase.

Taq polymerase was originally found in bacteria that live in hot springs. It is used because it does not denature at the high temperatures used in PCR and can therefore continue to function in repeated cycles.

First, the DNA is heated enough to break the hydrogen bonds that hold the two strands of the double helix together. This is called the denaturation phase. This occurs at a temperature of around 98 °C. Then, as the sample is allowed to cool to around 60 °C, the short primer sequences will bond to complementary sequences in the DNA sample. This is known as the annealing phase.

The final phase is the extension phase, which happens at about 72 °C. The bonding of primers allows Taq polymerase to replicate DNA using the primer as a starting point. (DNA polymerases are not able to add the first nucleotide of a DNA strand; they are only able to extend existing strands.) Once the DNA has been replicated, the DNA strands are heated to the point of separation and the process begins again.

Each time a cycle occurs, the amount of DNA doubles, resulting in exponential growth. Within a few hours, enough cycles of PCR have occurred to create billions of copies of the DNA sequence. This provides ample copies for gel electrophoresis and other tests.

Sometimes scientists need to know more about an individual’s DNA, but determining the sequence of the entire genome would be excessive. In these cases, a technique called gel electrophoresis can be used to identify some key features of the DNA. Gel electrophoresis uses an electrical current to move molecules through a semisolid medium or gel. The DNA molecules are separated by their size and amount of charge.

DNA molecules have a negative electrical charge and will move towards the positive electrode in an electric field. DNA molecules are often millions of base pairs long; too long to be separated by electrophoresis.

To get fragments of appropriate size, usually 250–30 000 base pairs (bp) in length, DNA is digested with special enzymes called restriction endonucleases. These enzymes cut the backbone of the DNA double helix at highly specific sequences, producing shorter DNA segments and distinctive fragment patterns (Figure 2). These patterns can be used to produce DNA profiles or DNA fingerprints, combinations of DNA sequences that are unique to each individual. This allows anyone (with the exception of identical twins) to be identified by their DNA.

Samples with fragments of DNA are loaded into small depressions, called wells, on one end of the gel (a jelly-like polymer). The gel is submerged in a buffer solution, and an electric current is run through the gel. The DNA samples must begin near the negative pole, so that they can spread out as they are drawn toward the positive pole

The gel is porous, and the DNA must travel through the spaces within the gel. Smaller pieces can slip through the spaces more easily, allowing them to travel further along the gel in a given amount of time. Usually, one or more of the wells is filled with a ‘DNA ladder’, which contains DNA fragments with a range of known lengths. By using the DNA ladder, the length of sample fragments can be determined.

Figure 4 shows a photograph of DNA fragments that have been separated within a gel. Because the DNA fragments themselves do not have a colour, the gel must be dyed. Ethidium bromide is commonly used because it binds to DNA and then fluoresces in ultraviolet light. Ethidium bromide binds strongly to DNA, so it produces a clear signal, but is mutagenic, so sometimes safer alternatives are used

Both PCR and gel electrophoresis have many applications. One example is DNA profiling, which can be used for paternity testing and in forensic investigations. DNA profiling is a technique that examines variable portions of DNA to create a profile or ‘fingerprint’ that is unique to the individual. Forensics is the use of science in criminal and legal cases, and DNA profiling is a powerful forensic tool. Each of our cells contains our entire genome, and we shed cells continually, leaving them in the environment around us. By analysing residue on a doorknob, a drinking glass, a piece of clothing and so on, we can determine if an individual’s DNA is present. For example, a blood stain on the suspect’s jacket that contains the victim’s DNA could be very powerful evidence.

Most genomes, including that of humans, have short, repeated DNA sequences called tandem repeats. The number of repeats can vary greatly between individuals in a population, meaning there may be dozens of versions. Restriction enzymes are used to chop the DNA into fragments that vary in length depending on the number of repeats. After amplifying with PCR, the resulting mix of DNA fragments is separated using gel electrophoresis

To match the DNA in a piece of evidence to the DNA of an individual, there must be exactly the same number and length of DNA fragments, since a person’s DNA sequence is the same in all cells

The nucleotides are held together by phosphodiester bonds. A phosphodiester bond occurs when two of the hydroxyl groups in phosphoric acid react with hydroxyl groups on other molecules to form two ester bonds. Phosphodiester bonds are central to all life on Earth, as they make up the backbone of the strands of nucleic acid and are the reason for its directionality. In DNA, the phosphodiester bonds occur between the phosphate group attached to the 5ʹ carbon of the deoxyribose of one nucleotide and the hydroxyl group on the 3ʹ carbon of deoxyribose on the next nucleotide. This arrangement allows us to describe the directionality in terms of 5ʹ and 3ʹ.

When assembling a new strand of DNA, DNA polymerase III adds the 5ʹ end of a DNA nucleotide to the 3ʹ end of the previously added nucleotide. Like other enzymes, DNA polymerase III has an active site that is complementary to only a very specific shape. This is why it is only able to build new DNA strands in a 5ʹ to 3ʹ direction. If it worked in the other direction, the shape would be different.

As new strands of DNA can only be assembled by DNA polymerase III in a 5ʹ to 3ʹ direction, only one strand can be replicated in the same direction as the helicase is unwinding and unzipping the original strand. As the helicase moves along the DNA, it forms the replication fork where replication can occur. The strand that can be replicated in the same direction as the helicase moves is called the leading strand. This strand is orientated from 3ʹ to 5ʹ and can be replicated continuously.

The other strand, as it is orientated in the opposite direction, does not allow DNA polymerase III to move in the same direction as helicase. It must work in the opposite direction. This strand is known as the lagging strand and the replication along this strand is discontinuous. DNA polymerase III replicates the new strand in sections, having to repeatedly move further along the strand to continue replicating it. These sections of newly formed but disconnected DNA are known as Okazaki fragments.

Several enzymes are involved in the process of DNA replication. These include DNA helicase, DNA polymerase (actually DNA polymerase III), DNA primase, DNA polymerase I and DNA ligase. As it is easier to understand, we will limit our understanding of these enzymes and how they function to the DNA replication within prokaryotic cells. The following outlines the functions of these enzymes.

Helicase – unwinds and unzips the DNA molecule by breaking the hydrogen bonds holding the complementary bases together. This forms the replication fork. The strands are then kept from coming back together by single strand binding proteins. These proteins attach to the single strands of DNA and prevent them from re-forming the hydrogen bonds between their complementary bases until replication can be carried out.

Gyrase – moves ahead of helicase, relieving the tension created by the unwinding and unzipping of the DNA double helix.

DNA primase – attaches small RNA primers, made of several RNA nucleotides, to the template strand. This allows DNA polymerase III to attach and begin assembling the free nucleotides into a new strand of DNA. Without the RNA primers, DNA polymerase III is unable to attach to the DNA strand properly. As replication is continuous on the leading strand, only a single primer is required. However, on the lagging strand, primers need to be placed at regular intervals to allow DNA polymerase III to attach at the multiple points necessary for the discontinuous replication on this strand.

DNA polymerase III – assembles the new strands of DNA by placing free nucleotides in the correct sequence according to the base sequence of the template strand and the complementary base pairing rule. It is only able to build new strands in the 5ʹ to 3ʹ direction. It can replicate continuously on the leading strand but must replicate the lagging strand discontinuously. This means it must replicate the strand in short sections called Okazaki fragments.

DNA polymerase I – removes the RNA nucleotides of the primers and replaces them with the correct DNA nucleotides.

DNA Ligase – catalyses the formation of the phosphodiester bonds between the Okazaki fragments. This makes the replicated strand built using the lagging strand into a single strand that can function normally.

DNA polymerase III has an additional, but essential, function in this process. It proofreads the newly formed DNA strand as it is being built (Figure 1). If a nucleotide is placed with a mismatched base, the incorrect nucleotide is removed and replaced with the correctly matching one. For example, if A is matched to C, the A would be recognised as being incorrect and it would be removed and replaced with G that is complementary to C. This is just one of several ways that cells are able to avoid the vast majority of errors that would lead to potential mutations.