MLAB 2337 Molecular Diagnostics Techniques

MLAB 2337 Molecular Diagnostics Techniques
Laboratory 7: Electrophoresis

OBJECTIVES

Briefly describe electrophoresis.
Describe the “sieving” affect.
State what agarose gel is made from, the concentration range most frequently used and the number of base pair fragments it is best at resolving.
State what polyacrylamide gel is compose of, the concentration and the number of base pair fragments it is best at resolving.
State the danger of preparing polyacrylamide gels.
List two of the most frequently employed stains used in electrophoresis.
State the potential hazard of using ethidium bromide.
State how ethidium bromide is used as a stain and how it must be read.
State how the Coomassie Brilliant Blue dye is used.
State the advantage of the BioRad Fast Blast DNA stain.
State the net charge of DNA.
Briefly state the principle of electrophoresis.
List the components of the TAE buffer and the purpose of each.
List the components of the TBE buffer and the purpose of each.
State the reason TBE buffer cannot be used for DNA which will be used in subsequent enzymatic reactions.
State the two functions of the loading dye.
List the 5(five) categories of potential sources of error and give one example of each.

INTRODUCTION

Electrophoresis is a method of separating molecules based on different physical characteristics, such as size and charge, by moving the molecules through a medium by applying an electrical current induced by electrodes. Agarose gel is one of the most frequently employed methods of separating and analyzing molecules, such as DNA, in biochemistry and molecular biology. When molecules are exposed to an electrical charge shorter molecules move faster and migrate farther than longer ones because shorter molecules migrate more easily through the pores of the gel matrix. The higher the concentration of the matrix the smaller the molecules which can pass through. This phenomenon is known as “sieving”. Knowing the size of the target molecules is crucial in selecting the concentration and type of gel to use for separating the target.

Gels

Proteins and nucleic acids are placed in a matrix or gel. The two most commonly utilized gels are agarose and polyacrylamide. Agarose gel is used to separate large nucleic acids (greater than a few hundred base pairs). Acrylamide is used to separate smaller nucleic acids, less than 500 bp.

Agarose is a polysaccharide extracted from seaweed. It is typically used at concentrations of 0.5 to 2%. The target molecule can be selectively separated by varying the concentration of the agarose, the higher the concentration of agarose the smaller the molecules that will be separated out. Fragments of DNA from about 200 to 50,000 bp can be separated using standard electrophoretic techniques. A 0.7% gel will show good separation of large DNA fragments (5-10 kb) and a 2% gel will show good resolution for small fragments (0.2-1kb).

Polyacrylamide is a cross-linked polymer of acrylamide. The length of the polymer chains is dictated by the concentration of acrylamide used, which is typically between 3.5 and 20%. Polyacrylamide gels are significantly more difficult to prepare than agarose gels because oxygen can inhibit the polymerization process. They must be poured between glass plates. Acrylamide is a potent neurotoxin and must be handled with care. Polyacrylamide is considered to be non-toxic but the gels should be handled with gloves due to the possible presence of free acrylamide. Polyacrylamide gels have a small range of separation but a very high resolving power. In the case of DNA these gels are used for separating fragments of less than 500 bp. Polyacrylamide gels are used extensively for separating and characterizing mixtures of proteins.

Most clinical laboratories purchase gels of the appropriate concentration to eliminate variables due to technique in gel preparation.

Dyes

Once electrophoresis is completed the bands must be analyzed. A variety of techniques and methods are available for detection of proteins in gels with the two most popular methods being ethidium bromide and Coomassie Brilliant Blue.

Ethidium bromide is added to the cooling agar before it is poured into the plates. Ethidium bromide is thought to act as a mutagen because it intercalates double stranded DNA (i.e., inserts itself between the strands), deforming the DNA. This dye is commonly used as a fluorescent tag. Ethidium bromide (EtBr) is a suspected carcinogen and should be handled with extreme care. Always wear gloves when handling the EtBr or the gels prepared with EtBr. The DNA fragments take up the dye as they migrate through the gel. Once the gel is run it is observed under ultraviolet light.

Coomassie Brilliant Blue nonspecifically stains proteins a strong blue color. The Coomassie dyes bind to proteins through ionic interactions between dye sulfonic acid groups and positive protein amine groups as well as through Van der Waals attractions. After electrophoresis the gels are removed from the buffer and placed in a solution of the Coomassie dye for a certain period of time. Several destaining steps are performed to finally visualize the bands, they are viewed with the naked eye. No special equipment is necessary to observe the bands which are formed. There are different formulations of the dye (R-250 and G-25) which have different applications based on the analysis performed.

Many other types of stains are utilized for staining gels. The BioRad Fast Blast DNA stain is a safe and non-toxic alternative to EtBr for the detection of DNA in agarose gels following electrophoresis. This stain contains a cationic compound that belongs to the thiazin family of dyes. The positively charged dye molecules are attracted to and bind to the negatively charged phosphate groups on DNA. The proprietary dye formula stains DNA deep blue in agarose gels.

PRINCIPLE

In simple terms electrophoresis is a procedure which enables the sorting of molecules based on size and charge. When charged molecules are exposed to an electric field they will migrate toward either the positive or negative pole according to their charge. In contrast to proteins, which can have either a net positive or negative charge, nucleic acids have a consistent negative charge imparted by their phosphate backbone and will migrate toward the anode (positive pole). The sample migrates through the gel in response to the electric current, the small molecules move more easily and more quickly than the larger molecules, which results in a distinct banded pattern forming in the gel. The bands are visualized using stains.

PROCEDURE

Performing electrophoresis requires the following:

1. Prepare samples, in this lab, DNA

2. Prepare buffer

3. Prepare gel

4. Add loading dye into sample

5. Load samples onto gel

6. Run gel in electrophoresis chamber for specified amount of time.

7. Stain, if necessary.

8. Interpret/analysis of gel

As part of the quality control in clinical laboratory testing a molecular weight standard and positive and negative controls are run in parallel with the patient samples. The molecular weight standard allows comparison of bands to the expected size. The controls are compared to the patient to determine if the target being analyzed is present or absent in the patient sample.

The preparation of the samples and reagents for the procedure is critical.

Sample Preparation – there must be enough DNA in the sample for a successful procedure. Most laboratories use kit tests with clearly defined procedures which, if followed exactly, should result in a sufficient quantity of product. If working in a research facility it is best to follow procedures to determine the quantity and quality of the DNA before proceeding.

Buffer – Buffers are used to make the gels and run the electrophoresis. In the electrophoresis chamber, the anode and cathode are separated and the gel is placed between them. In order to close the circuit and generate the voltage which causes the migration, the entire chamber is filled with a conductive buffer which allows the current to pass through. The electrophoresis buffer provides ions to carry a current and to maintain the pH at a relatively constant value. Buffers must be made up to the specifications of the procedure.

· Tris-acetate-EDTA (TAE) buffer is historically the most common buffer used for gel electrophoresis in the analyses of DNA products resulting from PCR amplification, DNA purification protocols or DNA cloning experiments. The Tris is useful in keeping the DNA deprotonated and in solution. Acetate gives improved separation of large DNA fragments. The EDTA protects nucleic acids from enzymatic degradation. TAE is the preferred buffer for isolating DNA fragments from gels to be used in subsequent enzymatic reactions. For example, if a DNA sample is going to be used for a cloning experiment TAE buffer is suitable for this purpose. TAE buffer provides faster electrophoretic migration of linear DNA and better resolution of supercoiled DNA.

· Tris-borate-EDTA (TBE) is also popular. TBE is better for isolating smaller DNA fragments. TBE buffers have a stronger buffering capacity for longer or higher voltage electrophoresis runs. For long runs TAE needs to be refreshed while TBE does not. Borate resolves fragments <2kb better. Borate is an enzyme inhibitor. This buffer cannot be used for DNA that will be used in subsequent enzymatic reactions.

Gels were discussed in the introduction. Select the type and concentration of gel suitable for the size of DNA fragments being tested for.

Samples – proper prepping of the samples is crucial. In DNA analysis the product is usually from a PCR. Errors in the PCR will translate directly into errors in results obtained unrelated to procedural errors in the electrophoresis. The correct amount of sample must be loaded into the appropriate well. It is crucial to properly identify each sample and the well it was added to.

Loading dye - Since DNA cannot be seen as it migrates through the gel a loading dye is added so that the movement of the DNA can be monitored. The loading dye serves two functions. The first function is weight. The loading dye is made with a high concentration of sugar which is heavier than the buffer solution in the tank. This causes the dye (and DNA contained within the dye) to sink to the bottom of the wells during the gel loading process. The second function is that it indicates when to stop the electrophoresis. The loading dye moves more quickly than the actual DNA. The power should be shut off before the dye reaches the end of the gel.

Electrophoresis chamber – the electrodes must be inserted properly. Most electrophoresis chambers have color coding to prevent set up errors. If the electrodes are inserted the wrong way the DNA will run backwards and off the gel. The voltage is crucial for a successful procedure. A higher voltage makes things move faster but may also end up degrading the sample or melting the gel. Follow the protocol exactly.

Staining – was discussed in the introduction. Select a staining method appropriate for the protocol performed.

Interpretation/Analysis – once bands are formed compare the patient samples and controls with the molecular weight standard to ensure the bands are of the correct size. Compare the patient with positive and negative controls to identify the products present.

Sources of Error

1. Sample contamination.

a. Take steps to prevent contamination by wearing gloves, using proper pipetting technique to prevent contamination of the pipette and changing tips after each use.

2. Buffer

a. Buffer was not diluted correctly or used the wrong concentration.

b. Buffer run to fast.

3. Gel

a. Make sure the concentration of gel is appropriate for the testing performed.

b. Accurately measure the buffer and agarose.

c. Pour gels so there are no bubbles or cuts.

d. Cool completely before adding samples.

4. Load appropriate amount of sample.

a. Too much of the target will cause a smear.

b. Too little of the target may not form a band or form a band that is difficult to see.

c. Load sample INTO the well. Take care not to get pipette tip at bottom as it will be dispensed under the well.

5. Electrophoresis

a. Current applied in wrong direction. Make sure the positive and negative are connected properly.

b. Too high a voltage may cause the sample to run off the gel or the heat produced may cause denaturation of the sample.

c. Too low a voltage will take too long to run.

d. Incorrect timing of procedure – too short separation will not occur, too long may run off the gel.

Assignments

· Review the “Gel Electrohoresis Virtual Lab” http://learn.genetics.utah.edu/content/labs/gel/

· Review “Gel Electrophoresis” from the DNA Learning Center: http://www.dnalc.org/resources/animations/gelelectrophoresis.html

· Review “Experiment 2: Gel Electrohoresis of DNA” virtual lab: http://www.life.illinois.edu/molbio/geldigest/electro.html

· Review “Separating Fragments of DNA by Gel Electrophoresis” http://www.sumanasinc.com/webcontent/animations/content/gelelectrophoresis.html

Name ______Date ______

MLAB 2337 Molecular Diagnostics Techniques
Laboratory 7: Electrophoresis

Study Questions

Points: _____ /32

Instructions:

· Copy/paste the following study questions into word processing document.

· Answer the questions

· Save the file as “Lab7Electrophoresis_YOURNAME”

· Submit to “Assignments” in BlackBoard

Describe the “sieving” affect. (1 point)
State what agarose gel is made from, the concentration range most frequently used and the number of base pair fragments it is best at resolving. (3 points)

State what polyacrylamide gel is compose of, the concentration and the number of base pair fragments it is best at resolving. (3 points)

State the danger of preparing polyacrylamide gels. (1 point)

5. State the process for utilizing ethidium bromide (EtBr) as a stain AND why it is considered dangerous. (2 points)

6. State how the EtBr “stains” the DNA AND how the bands are visualized. (2 points

State how the Coomassie Brilliant Blue works as a dye AND how the results are visualized (2 points)
State the advantage of the BioRad Fast Blast DNA stain.(1 point)

State the net charge of DNA. (1 point)
Briefly state the principle of electrophoresis. (2 points)
List the components of the TAE buffer and the purpose of each. (3 points)

List the components of the TBE buffer and the purpose of each. (3 points)

State the reason TBE buffer cannot be used for DNA which will be used in subsequent enzymatic reactions. (1 point)
State the two functions of the loading dye. (2 points)
List the 5(five) categories of potential sources of error and give one example of each. (5 points)

Lab 7: Electrophoresis Page 2