Chapter 12b Identification of Organisms Using 16SrRNA Sequencing, Session 2

.pdf

School

Temple University *

*We aren’t endorsed by this school

Course

3711

Subject

Biology

Date

Apr 3, 2024

Type

pdf

Pages

Uploaded by CorporalSparrowPerson1104

Chapter 12b: Identification of Organisms Using 16S rRNA Sequencing, Session 2 Objectives • To become familiar with DNA gel electrophoresis • To clean up and send out samples for sequencing DNA Gel Electrophoresis Electrophoresis is a method used to separate cell fractions, proteins, or nucleic acids by size and/or charge. There are many different protocols used in electrophoresis, but all take advantage of an inert support matrix (e.g., starch, agarose, polyacrylamide) and an electrical field. Molecules move in this electric field, but not all molecules will move at the same rate. The solid support that we will be using is agarose, which serves as a molecular sieve to separate the DNA molecules according to their size. DNA is negatively charged at neutral pH and thus moves towards the positive side of the gel. When mixed with water, boiled and then cooled, agarose will turn into a gel-like substance. This gel is a complex network of fibrils (of polysaccharide bonded together by hydrogen bonds) through which the DNA fragments must pass. The distance between the fibrils or the pore size is determined by the concentration of agarose used, i.e., a 20% agarose will hardly let very small DNA fragments to pass, while a 0.2% agarose is rather flimsy and can let very large DNA fragments travel through easily. So, choosing the appropriate agarose concentration is important. Today we will use a 1.0% gel. The shape of the molecules also plays an important role in relative migration distance in an electric field. Compact molecules move more rapidly than open molecules. For example, circular DNA that is twisted, or supercoiled, will migrate faster than open linear DNA. If you were to compare a supercoiled piece of DNA with a linear one of the same size, the supercoiled one would migrate farther on the gel. Today, you will be performing electrophoresis on DNA that is linear. Two other important factors are the amperage of the electrical field and the ionic strength of the electrophoresis buffer in which the gel is submerged. With higher amperage and/or buffers of high ionic strengths, the molecules move faster in the field. However, too high a voltage, amperage, or ionic strength of the buffer can cause heating of agarose and loss of its natural matrix and even denaturation of the DNA. Electrophoresis is conducted in a plexiglass apparatus composed of a middle section where the agarose gel is placed and two buffer compartments at either end, which are connected by platinum wires to cords that lead to a power supply. The researcher must take care not to inadvertently receive an electric shock.

Before loading the samples into the gel, we add a loading dye (bromophenol blue) to the samples. This is done for three reasons: it allows you to see the samples while you are loading the gel, and it allows you to track the progress of the gel as the samples migrate through it. Also, the loading dye also contains glycerol which helps draw the samples towards the bottom of the well during the loading process. Before the hot agar is poured into the tray to cool, we add an intercalating dye such as ethidium bromide (EtBr) or SYBR Gold. These are dyes that fluoresce under ultraviolet radiation and have a very high affinity for the double-stranded DNA molecule. Because EtBr and SYBR Gold intercalate with the DNA, only those areas of the gel that have DNA will fluoresce when exposed to UV light. We refer those glowing areas as “bands.” By comparing each band with a lane that contains DNA fragments of known size, called a ladder or standard, we can approximate the size of each fragment. The ladder that we will use is called GeneRuler 1 kb DNA Ladder. Below is what it should look like when run on a 1.0% gel. You will need this image to compare your own ladder to, so that you can estimate the size of the fragments that you amplified. Unfortunately, EtBr is a highly mutagenic and carcinogenic agent. Newer dyes have come to market that work well and are less toxic than EtBr. Since student safety is one of our priorities in this lab, we have replaced EtBr with SYBR Gold in all our exercises. However, it is more expensive, so be careful to pipet the correct amount. Although the Ames test has shown very low toxicity for SYBR Gold, you should still be cautious when using it, because any intercalating agent is, by nature, hazardous. The end goal of this exercise is to obtain the sequences of the 16S rRNA gene of your unknowns, so that you can determine their identities. The sequencing center charges $7.20 per reaction. It would be a waste of time to submit a tube which did not contain DNA. Therefore, we run a fraction of each PCR product on a gel to verify that the reaction worked. Recall that you set up two PCR reactions per unknown last week. You will submit for sequencing just one PCR product per unknown. The results of your gel will help you decide which of the two to submit. Think about what size band you expect to see on your gel.

ExoSAP-IT! (Cleaning Up PCR Product) ExoSAP-IT is a product that cleans up the PCR product, as shown in the illustration below. It consists of two hydrolytic enzymes: Exonuclease I, which digests the unused primers, and Shrimp Alkaline Phosphatase, which digests the dNTPs that remain in the PCR reaction tube. If left untreated, the primers and dNTPs would interfere with the sequencing reaction. These enzymes are active at 37°C but get deactivated at 80°C. After ExoSAP-IT treatment, the PCR product is ready to submit for sequencing.

Your preview ends here

Eager to read complete document? Join bartleby learn and gain access to the full version