Chemistry 301 Introduction to Biochemistry

Study Guide :: UNIT 9

Techniques

Overview

This unit will introduce some of the experimental procedures used in the study of biochemistry. Many of the techniques it will cover have been used to understand the concepts we have discussed in this course. As you read through the unit, you will note that experimental procedures have been developed based on knowledge that has been acquired over time about molecules and cellular processes. For example, we would not be able to utilize the polymerase chain reaction (PCR) in cloning without first understanding the role of DNA polymerase, DNA binding, and DNA replication. Please note not all techniques are covered in this lesson. Some important experimental procedures have been left out, such as NMR spectroscopy and X‑ray crystallography, which are used to determine molecular and protein structures respectively. This unit will provide information on techniques from the perspective of the whole cell down to the genetic level.

Unit 9 is divided into five lessons:

Learning Objectives

After completing this unit, you should be able to:

1. Discuss biochemical techniques and their applications.
2. Demonstrate an understanding of the methodology behind cell disruption and fractionation.
3. Demonstrate an understanding of the general methods of chromatography.
4. Demonstrate an understanding of the different forms of electrophoresis.
5. Differentiate between Southern, Northern, and Western blotting.
6. Outline the methods of microarrays, polymerase chain reaction (PCR), cloning, and reverse transcription.

Glossary

Lesson 1: Cell Disruption and Fractionation

Overview/Objectives

After completing this lesson, you should be able to:

1. Define the term “cell lysis.”
2. Describe methods of cell disruption.
3. Outline how cells are fractionated.
4. Define the term “centrifugation.”
5. Demonstrate an understanding of the purpose for cell disruption and fractionation to study cellular functions and processes.

Readings and Activities

1. In Chapter 9 Techniques, read “Introduction,” “Cell Disruption,” and “Fractionation” (pages 213–215 of the textbook).
2. You can also watch two video lectures on Techniques for all of the lessons in this unit:
   • #06 Biochemistry Protein Purification Lecture for Kevin Ahern’s BB 450/550

   • #07 Biochemistry Protein Characterization Lecture for Kevin Ahern’s BB 450/550

     (Links to these are also provided on page 213 of the textbook.)

Commentary

You can imagine how difficult it would be to study the mechanism of action of one enzyme, for example, in a cell or organism containing many different enzymes, not to mention all of the other structures and molecules present. It is therefore necessary to be able to isolate molecules (lipids, carbohydrates, DNA, and proteins) in order to study them individually. Cell disruption and fractionation are two widely used methods that allow biochemists and other scientists to separate and isolate the components they are interested in studying.

To isolate components, cells must first be disrupted. This is referred to as cell lysis, which means breaking the cells open by disrupting the membrane. One way to achieve this is to lower the ionic strength of the medium the cells are in. The lowering of ions in the medium causes an imbalance of ions between the inside and outside of the cell. To even the concentration of ions, water from the medium will rush into the cell (where the ion concentration is higher) by diffusion, causing the cells to burst open. Mild surfactants may be used to enhance the process.

Bacteria, yeast, and plant cells have cell wall structures that are more rigid and they are resistant to osmotic shock, so lowering the ionic strength of the medium is not effective. The following techniques may be used:

• Enzymes, lysozyme (for bacteria) and cellulase (for plants), are often used as they will degrade the cell wall.
• Mechanical agitation, with glass beads hitting the cell at high speed.
• Sonication employs sound waves to break cells open.
• Cell bomb: exposing cells to high pressure causes gases in the cell to be released as bubbles, which breaks the cells open.
• Cryopulverization: Tissues are flash frozen in liquid nitrogen and then ground to a powder. Useful for cells with a strong extracellular matrix such as connective tissue or seeds.

The point of cell disruption, no matter the method, is to break open cells and release the contents, which is the first step in being able to study a particular component in isolation. Cell disruption techniques result in what is called a “crude lysate”, meaning a mixed bag of molecules from lysed cells, and this must then be separated by fractionation to start to obtain a pure sample of a particular molecule.

The purpose of fractionation is to really separate the cell contents into subsets. This is achieved by centrifugation, which is spinning the samples at relatively high speeds in a machine called a centrifuge. Because structures with a higher mass will spin to the bottom of a tube more easily than smaller ones, you can begin to separate cell components based on the speed at which you spin the cells. The textbook does a good job of explaining how you might separate the nucleus from other smaller organelles. In terms of separating individual cell components, a series of centrifugations, as outlined in the figure on page 214 of the textbook, is performed to remove cell debris and isolate these components. In centrifugation, the portion of the sample that is spun down is referred to as the “pellet” and the liquid contents of the sample that remain are referred to as the “supernatant.”

In isolating whole cells, eukaryotic cells in media can be spun at lower speeds (approximately 2,000 rpm) as they are larger, whereas pelleting bacterial cells from a broth requires a higher setting (15,000 rpm) because they are much smaller. Separation of proteins can be achieved using different concentrations of glucose solutions in tubes to enhance separation by centrifugation (following several other steps). Genomic DNA isolation relies on a combination of steps and centrifugations to get the final product:

1. centrifugation of whole cells
2. cell lysis using a detergent
3. centrifugation to pellet/remove cell debris and cell wall material
4. precipitation of proteins from the supernatant
5. centrifugation to remove the protein pellet
6. precipitation of the supernatant with isopropyl alcohol to precipitate the DNA out of the liquid suspension
7. centrifugation to pellet the DNA

Centrifugation results in a subset of molecules. However, there are still many different molecules in each subset. In the case of proteins, it is very difficult to separate individual proteins from a mixture. That is the topic of the next lesson.

Study Questions

1. Explain the principle behind causing cell lysis by lowering the ionic strength of the media.
2. What is lysozyme? What does it do? Why is it used for bacterial cells?
3. Define the terms “centrifugation,” “pellet,” and “supernatant.”
4. How is centrifugation used to separate cellular components?

If you wish to discuss any of these questions or need assistance with the material, please contact your Academic Expert (AE) by emailing the Student Success Centre at fst_success@athabascau.ca.

Lesson 2: Chromatography

Overview/Objectives

After completing this lesson, you should be able to:

1. Discuss the purpose of chromatography.
2. Demonstrate an understanding of the different types of chromatography.
3. Describe at least one type of chromatography.
4. Demonstrate an understanding of the function of histidine tagging to purify proteins.

Readings and Activities

1. Read the last paragraph of “Fractionation” and read “Ion Exchange Chromatography,” “Gel Exclusion Chromatography,” “Affinity Chromatography,” “High Performance Liquid Chromatography (HPLC),” and “Histidine Tagging” (pages 215–218 of the textbook).
2. You can also watch two video lectures on Techniques for all of the lessons in this unit:
   • #06 Biochemistry Protein Purification Lecture for Kevin Ahern’s BB 450/550

   • #07 Biochemistry Protein Characterization Lecture for Kevin Ahern’s BB 450/550

     (Links to these are also provided on page 213 of the textbook.)

Commentary

As mentioned at the end of the last lesson, cell disruption and fractionation are the initial steps in separating components or molecules of cells, but these procedures result in subsets that still contain many different types of molecules. Chromatography is a technique that allows the separation of molecules to a higher resolution, and combined with other techniques, can allow the purification of one type of molecule, often one protein of interest.

Chromatography is often performed in columns, which are tubes containing material used for separation called a support. This support will have properties that can be used to purify the molecule based on size, charge, or other characteristics. The column is “packed” with the support and a buffer is added. The sample is added to the column and allowed to flow through the support; tubes of the different samples, or fractions, are collected as the sample flows through. As different molecules in the sample will interact differently with the support, the resulting fractions should have different molecules from the subset in them. The idea is that, if you are purifying one protein, that protein should be pure in one of the fractions. This lesson looks at several types of chromatography.

Ion Exchange Chromatography

In ion exchange chromatography, the support is composed of tiny beads with charged chemicals attached. Purification is dependent on the binding of the molecule of interest to the charged beads. Molecules in the sample that are neutral or the opposite charge will pass through the column and end up in the early fractions. The molecule of interest will bind the column and come out in later fractions when a counter‑ion is added to push it off of the column. Depending on the charge of the molecule to be purified, an anion or cation column is used. Molecules are separated based on their charge.

In cation exchange chromatography, the beads are covalently linked to a negatively charged chemical, and a positively charged ion is used as the counter‑ion. This is for purification of a molecule or molecules that are positively charged. When the sample is added to the column, the positively charged molecule of interest exchanges with the counter‑ion and binds the column. Negatively charged or neutral molecules pass through the column and come out in the early fractions or tubes. To remove the positively charged molecule of interest that is stuck to the column, a high concentration of the counter‑ion is added; this displaces the molecule of interest, and it flows through the column and comes out in later fractions. The figure on page 215 of the textbook illustrates cation exchange chromatography.

In anion exchange chromatography, the beads are covalently linked to a positively charged chemical, and so the counter‑ion used would be negatively charged as well as the molecule of interest. The negatively charged molecule or group of molecules present in the sample exchanges with the negative counter‑ion and binds the column. All neutral and positively charged molecules flow through quickly and end up in the early tubes or fractions. To remove the bound molecule or group of molecules, a high concentration of the negative counter‑ion is applied to the column and the “ion exchange” between the counter‑ion and the negatively charged bound molecules displaces the negatively charge molecules and they flow out of the column into the later tubes or fractions.

Ion exchange chromatography allows the recovery of all components of a mixture that have the same charge, and depending on the procedures completed before this step, it may allow for the purification of one protein. Knowledge of the relative charge of a protein is important to predict what type of column to use.

Gel Exclusion Chromatography

Gel exclusion chromatography (also called size exclusion, molecular exclusion, or gel filtration chromatography) is used to purify molecules based on their size. The column support is made of gel beads that have little tunnels or holes in them, which will allow only the passage of molecules of certain sizes. The size of the hole is called the “exclusion limit.”  Molecules that are larger than the exclusion limit in a mixture will not enter the tunnels but will pass through the column between the beads and flow out into the collection tubes or fractions early. Molecules that are the size of or smaller than the tunnels will flow through the column more slowly and elute in later fractions because they enter the tunnels. Therefore it is important if using this technique to have an idea of how large the molecule is you are trying to purify. The figure on page 216 of the textbook illustrates this form of chromatography.

Affinity Chromatography

Affinity chromatography is used to purify molecules, proteins in particular, by using a support that has a particular molecule covalently linked to the beads that the molecule or protein of interest has an “affinity” for and will bind. See page 217 of the texbook for a figure of affinity chromatography. The protein of interest can be released from the column by adding extra of the same molecule that is covalently linked to the support and will flow out in later fractions. Molecules are separated  by this method based on their binding affinity.

High Performance Liquid Chromatography (HPLC)

High performance liquid chromatography (HPLC), also called high pressure liquid chromatography, is used to purify or separate molecules based on their polarity. The supports are composed of tiny beads that are packed very tightly, so that high pressure is needed for solvents and buffers to flow through. The supports may be polar (normal phase separation) in which non‑polar molecules flow through followed by elution of polar molecules in later fractions; or non‑polar (reverse phase separation), where polar molecules are eluted first, followed by non‑polar molecules.

Histidine Columns

One of the approaches a scientist may take to study a particular protein, if they already know the gene sequence, is to have that protein cloned and expressed in a plasmid. We will cover cloning in another lesson, but the point of doing this is to have a pure protein with multiple copies to test. Once this protein is cloned into a plasmid and grown in a bacteria such as Escherichia coli, it is necessary to purify this protein from the bacterial cells. The best way to do this is to use histidine tagging and run the protein on a column. This is achieved by adding nucleotide sequences to your primers for cloning that after transcription and translation will result in six histidine residues added to the recombinant protein. After obtaining a cell lysate, the his‑tagged protein can be purified relatively easily by applying the sample to a protein with a support of cobalt or nickel, to which the histidine residues will bind. All other proteins in the lysate will not stick and will flow through the column. The his‑tagged protein can then be released from the support by adding imidazole to recover the pure protein from the column. The first figure on page 218 of the textbook illustrates histidine tagging and column recovery.

Study Questions

1. What is the basis for chromatography? What are the general components? How does it generally work? What is the purpose of this technique?
2. Say you are interested in purifying a molecule with a known negative charge. What type of ion exchange chromatography would you use? What would be the charge of the counter‑ion you would use?
3. What is a fraction? Do the molecules of interest come out in earlier or later fractions from an ion exchange column? Why?
4. What is the support made up of in gel exclusion chromatography? How does this allow separation of different‑sized molecules?
5. You have a protein of interest that you need to purify and have found that this protein binds strongly to GTP. What type of chromatography would be best to purify this protein? Why? How would you do this?
6. What molecular characteristics determine separation by HPLC?
7. What is a his‑tagged protein? How can this type of protein be purified?

If you wish to discuss any of these questions or need assistance with the material, please contact your Academic Expert (AE) by emailing the Student Success Centre at fst_success@athabascau.ca.

Lesson 3: Electrophoresis

Overview/Objectives

After completing this lesson, you should be able to:

1. Discuss the purpose of electrophoresis.
2. Describe the different methods of electrophoresis and identify how they are used.
3. Outline one application of electrophoresis.

Readings and Activities

1. Read “Electrophoresis,” “Agarose Gel Electrophoresis,” “SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE),” “Isoelectric Focusing,” “2D Gel Electrophoresis,” and “Protein Cleavage” (pages 218–222 of the textbook).
2. You can also watch two video lectures on Techniques for all of the lessons in this unit:
   • #06 Biochemistry Protein Purification Lecture for Kevin Ahern’s BB 450/550

   • #07 Biochemistry Protein Characterization Lecture for Kevin Ahern’s BB 450/550

     (Links to these are also provided on page 213 of the textbook.)

Commentary

The purpose of electrophoresis is to separate a mixture that is usually of the same type of molecule. Electrophoresis is used to separate DNA, RNA, and proteins based on their charge and or size. This involves applying the mixture or sample to a gel placed in an apparatus, covering the gel with buffer, and running electricity through the container, and hence through the gel and sample, for a period of time. The gel is then stained, depending on the type of electrophoresis, and the bands of individual molecules in the sample can be visualized.

Agarose Gel Electrophoresis

Agarose gel electrophoresis is used to separate nucleic acids. DNA and RNA are large molecules with an overall negative charge from the phosphate groups. The DNA or RNA is usually digested with restriction enzymes, which cut at particular sequences, before adding it to the gel. The amount of charge in DNA and RNA is proportional to the size of the fragments, and they can be separated based on size alone. Agarose is a gel‑like matrix with openings that allow molecules to pass. Once the electric current is applied to the gel encased in buffer and holding the DNA or RNA sample, the molecules start to move through the gel. Smaller fragments move faster, and larger fragments move slower through the gel. The gel is stopped by disconnecting the electricity, and is then stained with ethidium bromide, which intercalates into the DNA or RNA structure, allowing the bands to fluoresce under UV light.

File:DNAgel4wiki.png, Dr d12, 2006, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=1752980

If you are working with a particular fragment of DNA, the band can be visualized by agarose gel electrophoresis. If you cloned a particular gene into a plasmid and you wanted to check if that gene was ligated into the plasmid correctly, you could digest the plasmid DNA with restriction enzymes that you know will cut out the gene you cloned and then run the sample on a gel. Staining with ethidium bromide would reveal a larger DNA fragment and a smaller DNA fragment. The smaller DNA fragment would be the gene you cloned and the larger fragment the plasmid you cloned into. You would not be able to tell the sequence, but you could tell if the correct size DNA fragment was present. To confirm it was the right gene, you could do DNA sequencing to check that the sequence matched the DNA sequence of the gene you were trying to clone. Additional information on agarose gel electrophoresis can be found on the Wikipedia page Agarose gel electrophoresis.

SDS‑Polyacrylamide Gel Electrophoresis (SDS‑PAGE)

SDS‑PAGE is a form of electrophoresis designed to separate proteins. Proteins are large macromolecules too, but they are smaller than nucleotides and they vary in their charge. Because proteins are smaller, agarose cannot be used as the openings in the matrix are too large. The other important consideration with proteins is that they are globular. To separate proteins effectively, SDS‑PAGE uses acrylamide, a matrix that can be adjusted to separate larger or smaller proteins, and sodium dodecyl sulfate (SDS), a detergent that denatures the proteins and coats them with a negative charge so they can be separated by size. The SDS‑PAGE gel is run much like an agarose gel once the sample has been added, but with the application of an electrical current. Once the gel has been run, the proteins can be stained using Coomassie Brilliant Blue or silver nitrate for visualization. A molecular weight standard marker is run on the gel (Lane 1, on the left in the figure below) and stained as well.

Picture of SDS‑PAGE. Magnus Manske, 2003. CC BY‑SA 3.0. https://commons.wikimedia.org/wiki/File:SDS-PAGE.jpg

For more information on SDS‑PAGE, see the Wikimedia page Polyacrylamide gel electrophoresis.

Isoelectric Focusing

Isoelectric focusing is used to separate proteins based on their charge. Proteins vary in their charge and the pI of a protein is the pH at which its charge is zero. In order to separate proteins this way, a pH gradient is established in the acrylamide matrix. The sample is added, and an electric current is added. A protein with a positive charge will move through the gradient towards the negative electrode, but will stop at a region in the pH gradient where their charge is zero. At this point the protein is focused.

2D Gel Electrophoresis

2D gel electrophoresis combines the two techniques SDS‑PAGE and isoelectric focusing to allow the separation of proteins from a cell or tissue. After obtaining a cell or tissue sample, the cells are lysed and then subjected to isoelectric focusing. The sample is then separated using SDS‑PAGE. So, the proteins are separated first by charge and then by size. The result is in a gel with individual spots all around that correspond to a single protein. The spots can be cut out and sent for Mass Spectrometry analysis  to identify the individual protein. An example of a 2D gel electrophoresis is shown below.

Adapted from: Example of a 2D gel electrophoresis. From Tianjin Biochip Corporation, Proteomics, https://www.tjbiochip.com/templates/second-2/index.aspx?nodeid=204

As mentioned above, proteins that are identified by 2D gel electrophoresis in a sample may be subjected to mass spectrometry. Mass spectrometry can be used to determine a peptide sequence. In mass spectrometry, peptides are ionized and a plot of the masses of the fragments is generated. This can be used to decipher the peptide sequence.

Study Questions

1. What is the purpose of electrophoresis? What types of molecules are subjected to agarose gel electrophoresis? To SDS‑PAGE?
2. Why is agarose used in agarose gel electrophoresis, and acrylamide used in SDS‑PAGE?
3. What is the advantage of 2D gel electrophoresis?
4. You have a tissue sample of a wound and want to study what proteins are involved in wound healing. What type of electrophoresis would be best to identify one protein involved? What additional technique would be needed to identify that protein?

If you wish to discuss any of these questions or need assistance with the material, please contact your Academic Expert (AE) by emailing the Student Success Centre at fst_success@athabascau.ca.

Lesson 4: Blotting

Overview/Objectives

After completing this lesson, you should be able to:

1. Discuss the purpose of blotting.
2. Identify the different methods of blotting, and describe how they are used.
3. Outline one application of blotting.

Readings and Activities

1. Read “Blotting” (pages 223–224 of the textbook).
2. You can also watch two video lectures on Techniques for all of the lessons in this unit:
   • #06 Biochemistry Protein Purification Lecture for Kevin Ahern’s BB 450/550

   • #07 Biochemistry Protein Characterization Lecture for Kevin Ahern’s BB 450/550

     (Links to these are also provided on page 213 of the textbook.)

Commentary

The purpose of blotting is to identify a specific molecule from a mixture. This is achieved by using a probe or an antibody that will bind only that molecule. The mixture is first separated by gel electrophoresis (agarose gel for nucleic acids; SDS‑PAGE for proteins), and then the gel is blotted onto a membrane, meaning the nucleic acids or proteins are transferred onto a membrane or paper by an electric current so that they bind to the paper. After the molecules have been transferred, a probe is used that will bind the molecule of interest along with a compound that will allow visualization of the specific binding and identification of the molecule. There are three types of blotting experiments:

• Southern blot, used to identify individual DNA fragments or genes.

  The probe is a complementary sequence of DNA that will bind only the gene or fragment of interest. The probe is labelled with a dye or radioactivity so that the binding can be visualized. Example application: identification of a particular gene or lack of a particular gene due to a mutation in a human sample.

• Northern blot, used to identify an RNA sequence.

  The probe is a complementary sequence and labelled, similar to that for DNA. See page 223 of the textbook for a diagram of Northern blotting.

• Western blot, used to identify a specific protein.

  The probe used is an antibody that will bind the protein of interest. The antibody is then bound with a second antibody that is conjugated to a compound so that the binding can be visualized. This procedure can also be used to identify carbohydrates as they can be bound by specific antibodies too. Some excellent images demonstrating the full procedure can be found on the Wikipedia page Western blot. Example applications: diagnosis of HIV; presence of a bacterial capsular polysaccharide in clinical samples.

If you are interested, you may wish to look up the procedures in more detail, as they involve multiple steps; or look up some additional applications of these techniques.

Study Questions

1. What is the purpose of blotting? Differentiate between Southern, Northern, and Western blotting.
2. What is the typical probe used for Western blotting? How is the binding visualized?
3. Suppose you want to screen a patient for a mutation in the p53 gene, which is responsible for tumour suppression and control of malignant growth. Most people with cancer have a mutation in the p53 gene. What blotting method would you use to determine if a patient has a mutation in p53 and why?

If you wish to discuss any of these questions or need assistance with the material, please contact your Academic Expert (AE) by emailing the Student Success Centre at fst_success@athabascau.ca.

Lesson 5: DNA Techniques

Overview/Objectives

After completing this lesson, you should be able to:

1. Discuss the purpose of microarrays.
2. Outline the general procedure for making recombinant DNA.
3. Demonstrate an understanding of the theory and value behind the polymerase chain reaction (PCR).
4. Describe the uses of reporters.
5. Define “reverse transcription.”

Readings and Activities

1. Read “Microarrays” (pages 222–223 of the textbook); then read “Making Recombinant DNAs,” “Polymerase Chain Reaction (PCR),” “Lac Z Blue‑White Screening,” and “Reverse Transcription” (pages 224–227).
2. You can also watch two video lectures on Techniques for all of the lessons in this unit:
   • #06 Biochemistry Protein Purification Lecture for Kevin Ahern’s BB 450/550

   • #07 Biochemistry Protein Characterization Lecture for Kevin Ahern’s BB 450/550

     (Links to these are also provided on page 213 of the textbook.)

Commentary

DNA Microarrays

The textbook does a good job of explaining the theory and procedure behind DNA microarrays. This commentary will therefore focus on additional aspects of DNA microarrays and their applications. See page 222 of the textbook for an illustration of a microarray.

DNA microarrays provide an entire picture of what is going on in a certain condition by demonstrating what genes are expressed in a particular environment or tissue at a given time. They were considered to be very powerful when they were invented, because they were one of a few high‑throughput techniques that could generate a great deal of important data in one experiment. DNA microarrays are one of the technologies that has spurred the need and development of bioinformatics and other computing applications to analyze large amounts of data (big data). DNA microarrays are also referred to as “DNA chips.”

There are many applications of microarrays. The following table provides a list of applications. (It is not important to know all of these applications, but you should be able to cite one or two examples and overall demonstrate an understanding of the important contribution of this technology to science.)

Recombinant DNAs (rDNAs)

The ability to construct rDNAs is a technology that has changed the face of science and society in many ways. It has allowed researchers to determine the function of individual genes, to make mutations in genes and study the effect, to make compounds such as recombinant insulin (so that animals do not have to be sacrificed to produce insulin) for treatment of diabetes, and to engineer food to be more resistant to environmental conditions or pests.

The general procedure to construct rDNAs or to “clone” a gene is to obtain that gene and insert it into a plasmid, by digesting both the gene and the plasmid with restriction endonucleases or restriction enzymes that cut DNA at particular sequences. Insertion of the gene into the plasmid employs the use of the enzyme DNA ligase, which will allow the digested “sticky” or matching ends to join together. Bacterial plasmids are what makes cloning possible, as they contain features or are constructed to contain features that will allow production of multiple copies of a gene and a functional protein. The plasmid is transformed into a bacterium, such as Escherichia coli, and replicated. The gene is transcribed and a protein produced in multiple copies. Cells that contain the desired DNA or gene are identified, or selected, often because the plasmid carries an antibiotic resistance gene. These cells can be grown and the DNA isolated or the protein product purified.

The figure on page 224 of the textbook shows a typical plasmid. For a diagram of the process of making rDNA see the Wikipedia page Recombinant DNA.

Some specific examples of rDNAs developed are:

• Recombinant chymosin: Chymosin is an enzyme found in rennet that is needed to make cheese. It was the first genetically engineered food additive used commercially.

  Chymosin was formerly obtained from the fourth stomach of milk‑fed calves. Scientists engineered a non‑pathogenic strain of E. coli to be able to produce this enzyme in large quantities by cloning the gene into a plasmid capable of expressing the identical protein (enzyme) to be purified. The recombinant product costs less and is used in in about 60% of hard cheeses in the United States. It is considered safe for use by the FDA.

• Recombinant human insulin: This form has almost completely replaced insulin obtained from animal sources such as pigs and cattle for the treatment of insulin‑dependent diabetes. Recombinant insulin is made by inserting the human insulin gene into a plasmid for production in E. coli or yeast.
• Recombinant human growth hormone (HGH, somatotropin): This is used to treat patients who cannot generate sufficient quantities to support normal growth and development. Before rDNA technology was available, HGH was obtained from the pituitary glands of cadavers. Using HGH from cadavers led to some cases of Creutzfeldt‑Jakob disease, a prion disease (human version of mad cow disease). Recombinant HGH eliminated this problem. However, it is also now misused as a performance‑enhancing drug.

After learning the procedure for cloning, you wonder how the gene or DNA sequence is obtained in the first place. There are a couple of methods of obtaining the gene to work with. One of these involves cutting the gene out of a DNA library (which contains DNA fragments inserted into plasmids) using restriction enzymes that you know will cut in the right places. However, this involves first finding that gene, which in the past could be done by restriction mapping, cloning, and sequencing of fragments, as well as identifying the function of that gene with a number of tests. With all of the achievements in DNA sequencing of entire genomes, from bacteria to humans, and bioinformatics to annotate the information and predict function, getting that gene has become much easier for scientists. The best way to obtain a gene of interest is to make many copies of that gene based on the sequence from the genome; this is the topic of the next section.

Polymerase Chain Reaction (PCR)

PCR is another technology that has revolutionized science. It was invented by Kary Mullis, a brilliant scientist, in 1983. There are stories that Dr. Mullis came up with the idea for PCR while driving in his car at night in California. The idea was extraordinary, and Dr. Mullis received the Nobel Prize in Chemistry in 1993.

PCR is well explained in the textbook, and an illustration of this technique is on page 225. Essentially, PCR makes use of a process already at work in nature, DNA replication, to generate multiple copies of a gene or DNA sequence of interest, so that there is enough of that DNA to work with. Primers are designed complementary to the DNA sequence of interest and these are combined with dNTPs, DNA from the organism with the gene, and a heat resistant DNA polymerase. The tubes containing the PCR ingredients are put into a thermal cycler, which is a programmable machine that changes temperatures over a series of cycles. The changes in temperature allow the DNA to denature and open up, then allow binding of the primers (annealing), and then allow replication or elongation of the DNA using the DNA polymerase and the dNTPs.

The figure below shows the process with the temperature ranges that favour each step:

File:Polymerase chain reaction.svg, Enzoklop, 2014, CC BY‑SA 3.0 https://commons.wikimedia.org/w/index.php?curid=32003643

Applications of PCR include DNA cloning for expression or sequencing, DNA‑based phylogeny (functional analysis of genes), DNA fingerprinting (forensic science and paternity testing), diagnosis of hereditary diseases, and detection and diagnosis of infectious diseases.

Study Questions

1. What is the basic principle behind DNA microarrays?
2. Name and describe two applications of DNA microarrays.
3. What is the function of DNA ligase in constructing rDNAs?
4. Name one rDNA that is in use, and discuss why the rDNA form is beneficial.
5. What is the purpose of PCR?
6. Why does the DNA polymerase used in PCR need to be heat‑stable?
7. Name one application of PCR.
8. What is Lac Z Blue‑White screening used for?
9. What is the product of reverse transcription? What does the enzyme reverse transcriptase do?

If you wish to discuss any of these questions or need assistance with the material, please contact your Academic Expert (AE) by emailing the Student Success Centre at fst_success@athabascau.ca.

Answers to Unit 9 Study Questions

Lesson 1: Cell Disruption and Fractionation

1. Lowering the ionic strength of the media will cause cell lysis, because the water in the media will flow into the cell where the concentration of ions is higher in order to balance out the concentration. The rush of water into the cell by diffusion will cause the cell to burst.
2. Lysozyme is an enzyme that can break down bacterial cell walls. It is used because bacteria have cell walls that are resistant to lysing by just lowering the ionic strength of the media.
3. Centrifugation involves spinning of a sample at a high speed, which results in separation of cell components by density. A pellet is the part of the sample that is the sediment after centrifugation; the supernatant is the part of the sample that is the liquid at the top of the tube, or the remainder in the tube after the sample is pelleted.
4. Centrifugation is used to separate cellular components because spinning of cells at varying high speeds allows for separation of components by density or mass. Multiple centrifugation steps involving pipetting off the supernatant and re‑centrifugation will allow a differential separation of cellular components to a certain extent.

Lesson 2: Chromatography

1. Chromatography allows the separation of molecules to a higher resolution and may allow purification of one molecule such as a protein. It is performed in columns or tubes containing a material used for separation based on size, charge, polarity, or molecular interactions. A buffer is added, and the sample is then added and allowed to flow through the support matrix. Different tubes or fractions are collected as the sample flows through and one of the fractions should contain the purified molecule.
2. To purify a molecule with a known negative charge, you would need to use anion exchange chromatography. The charge of the counter ion would be negative.
3. A fraction is a sample that is collected in a tube as the sample solution is allowed to flow through the column. The molecule(s) of interest come out in later fractions from an ion exchange column because the molecule of interest would first stick to the column, allowing other components to flow through first and later be released after adding the counter‑ion to the column, which displaces the molecule of interest that is stuck to the column.
4. The support in gel exclusion chromatography is made of gel beads with small holes in them that allow only the passage of molecules of certain sizes. The size of the hole is called the “exclusion limit.” Molecules larger than the exclusion limit in a mixture will not enter the tunnels; they pass through the column between the beads and flow out into the collection tubes or fractions early. Molecules that are the size of the tunnels or smaller will flow through the column more slowley and elute in later fractions because they enter the tunnels. It is important, if using this technique, to have an idea of how large the molecule is you are trying to purify.
5. To purify a protein that binds strongly to GTP, you would use affinity chromatography because it will be easy to separate this protein from other components that do not bind specifically to GTP. You would use a column with GTP covalently linked to the support beads and add the sample. The sample will bind the column and all other molecules will flow through. You then remove or elute the protein by adding extra GTP in some media to the column, which will displace your protein and allow it to flow out.
6. The molecular characteristic that determines separation by HPLC, or high pressure liquid chromatography, is the polarity of the molecule you are trying to purify.
7. A his‑tagged protein is a recombinant protein that has a histadine tag attached to it to purify it on a column. This is achieved by adding the nucleotides needed for 6 histadine residues when cloning a gene into a plasmid. This type of protein can be easily purified using a histadine column, by applying the sample to a protein with a support of cobalt or nickel. The histadine residues will allow the protein of interest to bind the column, while all other proteins in the lysate will flow through. The his‑tagged protein can be released from the support by adding imidazole, which will displace the protein and allow it to flow through the column and be collected in a later fraction.

Lesson 3: Electrophoresis

1. The purpose of electrophoresis is to separate DNA, RNA, or proteins by size using an electrical current. DNA and RNA are separated by agarose gel electrophoresis, and proteins are separated using SDS‑PAGE.
2. Agarose is used in agarose gel electrophoresis, because agarose is a gel‑like matrix with openings that allow DNA and RNA to pass, and the openings in the matrix of agarose are too large to use for proteins. Proteins are smaller, globular, and vary in their charge, so acrylamide is used for SDS‑PAGE. Acrylamide is a matrix that can be adjusted to separate larger or smaller proteins, and sodium dodecyl sulfate (SDS), is a detergent that denatures the proteins and coats them with a negative charge so they can be separate by size.
3. The advantage of 2D gel electrophoresis is that proteins can be separated by charge and size. This provides more separation of a mixture of proteins, so that each spot on the gel should be only one unique protein. The spots can be cut out and sent for Mass Spectrometry analysis to identify the individual protein.
4. The best method to identify a protein involved in wound healing would be to use 2D gel electrophoresis, so that you can separate individual proteins. Mass spectrometry would also be needed to find the sequence and identity of the protein.

Lesson 4: Blotting

1. The purpose of blotting is to identify one DNA or RNA sequence or one protein from a mixture. This is achieved by using a probe or an antibody that will bind only that molecule. Southern blots are used for DNA, Northern blots for RNA, and Western blots for protein.
2. The typical probe used for Western blotting is an antibody specific to the protein of interest. The antibody is bound with a second antibody that is conjugated to a compound, so that the binding can be visualized.
3. To determine if a patient has a mutation in the p53 gene, you would use Southern blotting, because this is the method used for identifying a DNA sequence of interest or a change in a DNA sequence.

Lesson 5: DNA Techniques

1. The basic principle behind DNA microarrays is that they can provide a lot of information about what genes are being transcribed in a cell under a particular condition. DNA microarrays provide an entire picture of what is going on in a certain condition by demonstrating what genes are expressed in a particular environment or tissue at a given time.
2. Two applications of DNA microarrays are:
   • Gene expression profiling: expression levels of thousands of genes are simultaneously monitored to study the effects of treatments, diseases, developmental stages.
   • Comparative genomic hybridization: compares the genomes of different cells or closely related organisms to see if there are differences in what genes they possess.
3. DNA ligase is used in the construction of rDNAs to join the digested “sticky” or matching ends together so the DNA sequence of interest can be inserted into a plasmid.
4. One rDNA in use is recombinant human insulin. The recombinant form is beneficial, because animals do not have to be used as a source of insulin for treatment of insulin‑dependent diabetes. The recombinant form is a harmless way of having abundant insulin available at a lower cost.
5. The purpose of PCR is to obtain multiple (millions) of copies of a gene or a DNA sequence, so that it can be visualized or used for cloning.
6. The DNA polymerase used in PCR has to be heat‑stable, because the high temperature used to separate DNA strands during PCR so that they can be replicated can cause an enzyme to degrade.
7. One application of PCR is DNA cloning for expression or sequencing. Others are DNA‑based phylogeny (functional analysis of genes), DNA fingerprinting (forensic science and paternity testing), diagnosis of hereditary diseases, and detection and diagnosis of infectious diseases).
8. Lac Z Blue‑White screening is used to identify clones that contain the correct DNA sequence inserted into a plasmid. This is because, when the DNA is cloned into a plasmid containing the β‑galactosidase gene, properly adding the transformed bacteria with the plasmid to media containing X‑gal will allow identification of the clones with the properly inserted DNA sequence, because those clones will be able to utilize the lactose in the agar plate and the colony will turn blue.
9. The product of reverse transcription is a cDNA or complementary DNA sequence. The enzyme reverse transcriptase converts RNA to a DNA sequence.

If you wish to discuss any of these questions or need assistance with the material, please contact your Academic Expert (AE) by emailing the Student Success Centre at fst_success@athabascau.ca.