Course: SAT 170 Introductory Biotechnology

Unit 9 Learning Module

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DNA Technology Part 1 (continued)

Figure 5

Figure 6

Gene Cloning
Being able to make several copies of a protein or piece of DNA is an important part of biotechnology. We discussed in our last unit using various host cells to produce a particular protein in a process we described as upstream processing. In this unit we will focus on making copies or clones of a particular piece of DNA, a process often referred to as gene cloning.  Gene cloning is a type of molecular cloning.<br /><br />The primary mechanism by which we make clones of a particular piece of DNA is known as Polymerase Chain Reaction (PCR). The requirements for such a reaction are as follows: a DNA template that is to be copied, a heat stable DNA polymerase molecule, a set of primers, a pool of nucleotides and a thermal cycler.<br /><br /><em>See Figure 5: All of the components of the polymerase chain reaction, including the heat stable DNA polymerase, nucleotides, primers and target DNA to be copied, are added to a small reaction tube such as the ones seen here.<br /><br /><em>See Figure 6: </em>The PCR tubes are then placed in a thermal cycler such as the one shown here.<br /></em>To understand PCR, you need to be familiar with the process of DNA replication as it naturally occurs in the cell. Recall from unit 3 that during DNA replication an enzyme known as helicase comes in and unwinds or separates the two strands of a DNA molecule. DNA polymerase is the enzyme responsible for using these separated strands to create complementary daughter strands. Recall that DNA polymerase relies on primers to get this process started. The PCR takes advantage of this natural process and so it requires primers and a DNA polymerase to be added to the reaction tube. Instead of using an enzyme like helicase, however, PCR uses heat to separate the two strands of a DNA molecule in a process known as DNA melting or denaturation. Recall from unit 3, that these two strands of DNA are held together by hydrogen bonds. These bonds are relatively weak, so the application of heat causes these bonds to break, thus separating the strands. The temperature is then decreased to give the complementary primers provided in the reaction a chance to anneal (bind to) the separated strands. The primers are provided in a much higher concentration than the original sample of DNA, so the strands are more likely to attach to a primer than they are to attach back to one another. This is known as the annealing step of the reaction. Once the primers are in place, the DNA polymerase is able to use the nucleotides provided as part of the reaction to build the complementary strands. This is known as the elongation step of the reaction. This process can be repeated several times, with the number of copies of the DNA doubling with each round of replication. The temperature fluctuations are essential to the reaction. This is the purpose of the thermal cycler, which is a machine that is capable of creating these changing temperatures within the reaction chamber. To ensure that the DNA polymerase does not denature during the process (recall from Unit 5 that extreme temperatures can cause proteins to lose their three-dimensional functional shape in the process of protein denaturation), a heat stable DNA polymerase is selected for the reaction. The most commonly used DNA polymerase for PCR is known as Taq polymerase. This enzyme was isolated from a thermophilic species of bacteria that inhabits environments such as hot springs. Thermophiles are known to thrive at relatively high temperature (temperature well over 100°F, often up to 250°F) which explains why Taq polymerase can tolerate the extreme heat needed to separate the strands of DNA during DNA melting.<br /><br /><em>See Figure 7: This diagram illustrates the steps of the polymerase chain reaction (PCR).</em><br /><br />

Figure 7

Figure 8

DNA Sequencing

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Figure 10

The process of DNA sequencing has provided valuable insight to the genome of many different organisms. DNA sequencing is performed in order to determine the specific sequence of nucleotides in a given sample of DNA. The techniques used for DNA sequencing have changed significantly, making it relatively easy to now sequence an entire organism’s genome. With this ability, the applications of DNA sequencing have also grown. DNA sequencing can be used to compare the evolutionary relationships between different species. It can also be used to identify certain genes that could be linked to certain types of diseases or a predisposition to certain types of cancers.<br /><br />One of the earliest and most widely used methods for DNA sequencing was developed in the 1970’s by Frederick Sanger. This method is commonly known as the Sanger Method.<br /><br /><em>See Figure 8: Frederick Sanger was awarded the Nobel Prize in 1980 for his work on DNA sequencing.</em>Since its introduction, the Sanger Method has been refined and optimized but it is still based on the use of dideoxynucleotide triphosphaste (ddNTP) molecules and a form of sequencing known as the chain termination method. A ddNTP is a type modified nucleotide which has hydrogen rather than a hydroxyl group at the 3’ end of the deoxyribose sugar. Recall from unit 3 that new nucleotides are only able to attach to the 3’ end of a growing DNA strand. The reason that the ddNTP modification is significant is that it prevents the addition of additional nucleotides. Thus, once a ddNTP is incorporated to a growing chain, the chain stops growing, or terminates. The sequencing method takes advantage of this by incorporating different types of labeled ddNTP’s for adenine (ddATP), guanine (ddGTP), cytosine (ddCTP) and thymine (ddTTP).  Each of the different ddNTP’s will give a slightly different signal, or fluorescence, allowing you to differentiate them from one another. Imagine these as different colors where ddATP will be red, ddGTP will be green, ddTTP will be black and ddCTP will be blue. The sequencing reaction is set-up much like a typical PCR, where you have a DNA polymerase, primers and normal deoxyribose nucleotides (dNTP’s), but you will also add the differently labeled ddNTP’s. Thus, if in the resulting products you get a segment that is 5 nucleotides long and is giving off a black color, than you know that the 5<sup>th</sup> nucleotide was T. If you line up all of the possible fragments based on their size you can get a complete sequence of that DNA sample. For example, if you had a segment that is 4 nucleotides long and is red, a segment that is 3 nucleotides and is green, a segment that is 2 nucleotides and is blue, and a segment that is 1 nucleotide and is black; you know that the complete sequence would be TCGAT.<br /><br /><em>See Figure 9: Notice how the addition of the different types of di-deoxyribonucleotides (ddNTP’s) results in the termination of a complementary strand creating different sized fragments in the sequencing reaction. In the original Sanger Method, these fragments would then be separated using electrophoresis as shown here.</em>The original Sanger method of DNA sequencing relied on electrophoresis for the separation of the different sized fragments before the fragments could be evaluated by their particular type of fluorescence.<br /><br /><em>See Figure 10: Using the original Sanger Method of DNA sequencing, each type of ddNTP would be run in different reactions and then separated in different lanes of the agarose gel. One could examine the fragment sizes and measure the signals of each ddNTP to determine the sequence of a particular DNA sample.</em>The original Sanger Method proved to be time consuming and thus limited the amount of DNA that could be processed in a short period of time. Computer-Automated or High-Throughput Sequencing eventually replaced the original Sanger method by using a small capillary gel to separate the different sized fragments. The gel can then be scanned with a laser to detect the fluorescence of each fragment and thus determine the terminal nucleotide of that fragment. This data is collected by a computer for processing and a print out of the sequence is generated based on the fluorescence pattern and its correlation to fragment length.<br /><br /><em>See Figure 11: This diagram illustrated the process of using a computer and capillary gel to automate the process of DNA sequencing. Such techniques are referred to as Computer-automated or High throughput sequencing.</em>In response to the demand for producing DNA sequences as a much faster rate, Next Generation Sequencing (NGS) techniques are continually being developed. These new sequencing methods are able to turn out more sequences in a shorter period of time, because they run multiple reactions in parallel to collect more data in a single sequencing run. Although there is a significant investment in the start-up costs to procure the equipment and training needed for the NGS techniques, they are still able to produce DNA sequences at a fraction of the cost compared to older methods.<br /><br />The human genome project is perhaps one of the largest research endeavors to employ the technique of DNA sequencing. Recall from unit 1, that the human genome project began in 1990 when groups of researchers decided to determine the entire sequence of nucleotides in the human genome. The final sequencing map was published in April of 2003. Since that time, this map has provided researchers with loads of information about human heredity and disease.

Figure 11