Chapter 11. LAB 11 Molecular Techniques II: Fingerprinting DNA

Learning Goals:

• Know what is meant by a DNA fingerprint and how DNA fingerprints can be produced through restriction enzyme digestions.
• Understand how agarose gel electrophoresis allows for the resolution of DNA fragments of differing sizes.
• Know the steps necessary to solve the case of the alleged “nosocomial infection” using DNA fingerprinting.
• Know how to retrieve nucleotide sequences from the NCBI GenBank database and use scientific software, such as NEBcutter, to produce DNA fingerprints made by specific restriction enzymes.
• Understand how to read a gel picture of DNA fragments and estimate fragment length using a DNA marker and migration distances.

Scientific Inquiry

What do CSI, Bones, and NCIS have in common? They are television series that showcase forensic science—a field of science that uses many different scientific techniques to investigate legal issues. The popular media has capitalized on the wonders of forensic methods ever since DNA fingerprinting became an important source of court evidence in the early 1980s. Although forensic science itself is a rather old discipline (predating popular characters such as Sherlock Holmes), it was not until the dawn of DNA technology that it became a mainstream topic.

DNA fingerprinting was invented by a geneticist, Dr. Alex Jeffreys. In the late 1970s, Dr. Jeffreys was interested in detecting variations within human DNA. He began with the β-globin gene family because it was one of the few human genes characterized at the time. Now, about 35 years later, every human gene has been sequenced and mapped to a particular chromosome, but when Dr. Jeffreys started his work, very little was known about DNA sequences.

Dr. Jeffreys used restriction endonucleases (enzymes that cut DNA) and agarose gel electrophoresis to show variations in DNA lengths called restriction fragment length polymorphisms (RFLPs) at the β-globin locus (Jeffreys 2005). Some alleles of genes at the β-globin locus gave one pattern of DNA fragments, while other alleles from the same locus gave a slightly different pattern, due to changes in the size of the restriction fragments produced (Figure 1). However, these RFLPs did not work well for characterizing differences in alleles between humans.

This led to the search for and discovery of genetic markers called minisatellite DNA. Minisatellites are short stretches of a repeating sequence (usually 10–30 base pairs) that occur in tandem (one right after the other) in many regions of the human genome (Figure 2). While the number of these repeats is highly variable, this variability is inheritable so that family members can easily be distinguished from non-family members. The pattern of these minisatellites that is produced after restriction digestion and agarose gel electrophoresis allows one to distinguish different individuals. Repeat, the pattern of these minisatellites produced after restriction digestion and agarose gel electrophoresis allows one to distinguish different individuals,using DNA samples only. The implications of this discovery were staggering.

The first test of this new technology involved an immigration case. A lawyer requested Dr. Jeffreys’ help in providing evidence that a young man from Africa was actually a woman’s son so that he would not be deported. Traditional blood tests showed that the woman and the boy were related, but could not show how closely they were related. Dr. Jeffreys was able to use these minisatellite markers and show that the woman was significantly more related to the boy than an aunt would be and the boy retained his citizenship (Jeffreys 2005). Subsequently, Dr. Jeffreys solved a double murder rape case in Leicestershire, England. DNA samples were collected from men in all the local pubs in the area. The original suspect was released because there was no match between his DNA and the DNA from the semen left at the scene. This was the first time that DNA was used to vindicate someone. The ability of DNA evidence to exonerate an innocent person spurred the formation of the Innocence Project in the United States in the 1990s.

Although DNA fingerprinting has advanced considerably through the invention of PCR and development of better DNA markers, the simple process of isolating DNA and mapping it with restriction enzymes and agarose gel electrophoresis that Dr. Jeffreys discovered is not outdated. These techniques are still commonly used even in the most sophisticated of molecular biology laboratories and they are still used forensically.

For example, if a bacterial infection breaks out at a hospital, and if the infection confers resistance to a particular antibiotic, it is most likely due to plasmid exchange between bacteria, since the plasmid usually contains genes that confer antibiotic resistance for its host bacteria. The bacteria are cultured and the plasmid DNA is isolated and digested with restriction enzymes to produce a plasmid fingerprint (Figure 3). If all of the plasmids isolated from the different infections produce the same restriction pattern, then the infection most likely originated from the same source and the origin of this source can be tracked with more advanced methods.

During this lab, you will work to solve a fictitious case using the same forensic analysis techniques that scientists use. You will also learn molecular biology approaches used to analyze recombinant DNA molecules. To this end, you will start with small rings of DNA called plasmids, which have already been isolated from bacterial cells. Plasmids are vectors commonly used for cloning genes, and most cloning procedures use the bacterium E. coli. A second important procedure in this technology is the digestion of plasmid DNA into manageable pieces with the use of restriction enzymes. Once your plasmid DNA is cut, you will size sort the fragments using gel electrophoresis and actually see the fragments in the gel using special stains. Gel electrophoresis allows you to sort DNA molecules by size and see the fragment patterns produced by restriction enzyme digestion.

Although plasmid vectors can be a source of DNA for forensic analysis, these rings of DNA can also be cut open with restriction enzymes, fused with a foreign piece of DNA and re-sealed with ligase, recreating a new and larger plasmid ring of recombinant DNA. This is gene-splicing, a technique for introducing new genes into a plasmid so the gene product can be produced by a host bacterium. Recombinant DNA molecules are inserted into a new host cell (usually E. coli) in a process called transformation. Transformation in a lab is the same process that occurs naturally in bacteria in the wild, plasmids are transferred from one bacterium to another with their genes, which often contain genes for antibiotic resistance.

In this lab, you will become a forensic scientist and provide information that will aide in a case of alleged harm due to a hospital-induced, or nosocomial, infection (Singh et al., 2006). The lawyer for the defendants states that their young daughter entered the hospital for a relatively simple operation. Her stay was prolonged due to the development of a bacterial infection that caused grave complications and required an additional surgery that was only partially covered by her parents’ insurance. The hospital is the plaintiff in this case, demanding full payment for the additional surgery. The parents believe the infection was nosocomial and report that there was a rash of such occurrences during their daughter’s stay. They report that everyone on their daughter’s floor had to gown up and wear masks before they entered this particular hospital ward. The hospital claims that the young girl’s infection is not related to that of any of the other patients and the mask and gowns were a precaution to prevent any spread of resistant bacteria.

Four different patients (A, B, C, and D) from her ward volunteered to be tested to determine if there was a common source of infection at the hospital. Your group will complete the forensic analyses for two of these samples (A&B, A&C, A&D, B&C, B&D, or C&D) to determine whether the plasmids isolated from the infectious bacteria is the same as the plasmids isolated from the infectious bacteria that led to the young girl’s complications. If your team can show that there is at least one other patient match, the court will mandate testing of samples from all the patients on that hospital ward.

Background

Restriction Enzymes

Restriction enzymes, also known as restriction endonucleases, catalyze double-stranded breaks in DNA molecules. You can think of them as very specific “DNA scissors” where each restriction enzyme binds to specific “recognition sequences” of nucleotides at cleavage sites. The recognition sequence for each restriction enzyme is palindromic, which means that it reads the same forward as it does backwards. Since DNA is double stranded, the sequence reads 5’ to 3’ on the top strand the same as it does 5’ to 3’ on the bottom, or complementary, strand. For example, the recognition sequence for the restriction enzyme PvuII is CAGCTG (Figure 4).

It reads CAGCTG going 5’ to 3’ on the top DNA strand and it reads CAGCTG when going 5’ to 3’ on the bottom DNA strand.

When PvuII cuts DNA, it cuts straight across from one DNA strand to the other, thus it makes a blunt cut. It cuts the phosphodiester bond between the G and the C going 5’ to 3’ on the top DNA strand and between the G and the C on the complementary strand. Not all restriction enzymes cut DNA this way. Some, such as BamHI with the recognition sequence G^GATCC, make staggered cuts in the DNA leaving what are known as sticky ends (Figure 5).

Any DNA molecule cut with BamHI can be joined together with any other DNA molecule cut with BamHI because the bases at the end of each cut will pair with each other. Restriction enzymes that leave cohesive, or sticky ends are restricted to pairing sticky ends with matching base pairs, whereas blunt ends cut with enzymes, such as PvuII, can match to any other DNA fragment with a blunt end, regardless of the enzyme that cut it. So, while ends produced by the restriction endonuclease BamHI are not compatible with ends produced by that of HindIII (another restriction enzyme that leaves sticky ends), ends produced by the two blunt cutters PvuII and EcoRV are compatible. The covalent joining of two different DNA molecules into a single continuous DNA molecule is the basis for recombinant DNA or cloning technology.

Agarose Gel Electrophoresis

DNA samples that have been digested using restriction enzymes usually contain a mixture of many billions of DNA fragments of different sizes. These fragments can be separated by size using a technique known as agarose gel electrophoresis, which not only separates DNA fragments by size, but also allows their visualization with the use of special dyes (Figure 6). Gel electrophoresis exploits the fact that DNA is negatively charged due to its phosphate groups—therefore DNA will naturally be drawn towards the positive pole in an electric field. The agarose gel is a porous, semi-solid that acts as a selective molecular sieve allowing smaller DNA fragments to pass through it more quickly than larger DNA fragments.

DNA samples mixed with loading dye are added to the wells at one end of the gel. An electrical current is applied across the gel, which drives the negatively charged DNA fragments away from the negatively charged cathode end of the gel towards the positively charged anode end of the gel—the shorter DNA fragments travel farther than the longer fragments.

In preparation for electrophoresis, the agarose gel is immersed in a buffer. Loading dye is added to the DNA samples before they are injected into the wells of the agarose gel. Loading dye contains a viscous agent that keeps the DNA sample in the gel wells so it does not float out into the buffer. The loading dye also contains colored dyes that migrate similarly to certain DNA fragment sizes so we can tell how far the invisible DNA has travelled through the gel. When a voltage current is applied across the gel, the DNA fragments migrate through it from the negative towards the positive electrode and sort by size as they go. The longer (larger) DNA fragments reptate, or “snake,” through the porous gel material more slowly than shorter fragments.

DNA itself is not visible during the gel run unless a fluorescent dye has been added to the agarose gel during its preparation and the gel is exposed to UV light. These dyes “squeeze” between the DNA bases and fluoresce green, orange, or gold when exposed to UV light depending on the type of dye used. The pattern of sorted DNA fragments on your gel should correspond to the DNA fragment sizes predicted by your restriction digestion calculations. You can record your results by taking pictures of the DNA fragments with conventional Polaroid photography or by digital imaging. One lane of your gel will contain a DNA marker that is made up of a mixture of DNA fragments of known sizes. The size of each unknown DNA fragment can be estimated by comparing it to the known sizes of the fragments in the DNA marker run in an adjacent lane of the gel (Figure 7).

You will use restriction enzyme analysis to analyze your plasmids isolated from the bacterial infections of patients A, B, C, and D and determine if they produce restriction patterns identical to that of the plasmid DNA sample from the bacteria isolated from the defendant. Would you expect identical DNA molecules to produce the same restriction map for a given restriction enzyme?

Resources

11.1 Use of Software in Science

(Complete this assignment prior to lab)

Learning Objectives

After successful completion of this activity, you should be able to:

Use of scientific software programs for planning experiments and analyzing data is integral to the modern research process. If you plan to clone a gene, there are programs to help you determine which restriction enzymes would be best to use. But you need to know 1) the sequence of the gene and 2) the sequence of the plasmid that you plan to insert the gene into. You may be able to retrieve the gene sequence from the public government database at NCBI called GenBank. If the plasmid was purchased from a biological company, then you can probably copy and paste the sequence from their website.

Once you know the sequence of your DNA, you can use NEBcutter, software that is freely accessible on the Web, to produce a restriction map of your DNA. A restriction map is a list of every enzyme that you designate and the exact position it cuts your DNA. If you know the GenBank accession number for your DNA (this is the number whereby every DNA molecule in GenBank is cataloged), then NEBcutter will retrieve the DNA sequence for you. If you are interested in the restriction map of a particular piece of DNA for a given enzyme, then this software is an easy way to produce a virtual gel run of that result.

Prepare for this lab by performing a virtual digestion and agarose gel electrophoresis of a plasmid called pMC524/ MBM. This plasmid was isolated during the study of multi-drug resistant nosocomial Staphylococcus aureus (Bhakta and Bal 2003). This plasmid also replicates in E. coli.

Activity 1: Restriction Digestion of Miniprep DNA

Learning Objectives

After successful completion of this activity, you should be able to:

• Restriction digest DNA samples and diagram the process of DNA digestion of a plasmid or linear DNA molecule (LO116)

Procedure

1. Check that the water bath temperature is 37°C—the optimal temperature for the enzymes you will be using in today’s lab.
2. Get your assigned plasmid DNA samples from your instructor. Thaw the samples and spin them briefly in the microcentrifuge to bring all the droplets to the bottom of the tube. If the volume of the DNA is less than 30 μl, see your instructor. Place the DNA samples on ice.
3. Two group members will prepare the restriction digestions while the other two group members set up the prepoured agarose gel in the gel tank (Activity 2). Your lab instructor will prepare the defendant plasmid DNA.
4. Label four 500 μl microcentrifuge tubes 1E, 1P, 2E, and 2P in which P stands for PvuII and E for EcoRI. Use one color tube for the PvuII digestions and the other color tube for the EcoRI digestions.
5. Set up your restriction enzyme reactions with PvuII first according to the table. Please note that you are digesting plasmids isolated from bacterial samples from two of the four A, B, C, or D patients (A&B, A&C, A&D, B&C, B&D, or C&D). Each plasmid DNA will be digested with PvuII and EcoRI. Write in the table under Tube 1P and Tube 2P the corresponding patient sample letter (A, B, C, or D).

6. Mix the contents by pumping the mixture up and down with the pipettor a few times. Shake the contents to the bottom of the tube, or spin briefly in the microcentrifuge using an adaptor tube. Will your DNA samples digest properly if some of this reaction mixture sticks to the sides of the tube?
7. Place your two PvuII reaction tubes in the 37°C water bath (Figure 9) immediately. Do not wait until your EcoRI digestions are ready to incubate. Thirty minutes is the minimal time needed to incubate your samples and it is best if the PvuII samples incubate longer than the EcoRI samples.
8. Prepare your enzymatic reactions for digestion with EcoRI according to the table.
9. Mix the contents by pipetting up and down a few times. Shake the contents to the bottom of the tube or spin briefly in the microcentrifuge using an adaptor. Place your EcoRI reaction tubes in the 37°C water bath.
10. Set your timer for 30 minutes. You will remove the PvuII samples and EcoRI samples at the same time.
11. While your DNA samples are digesting, prepare your uncut DNA control samples. Label two tubes 1U and 2U for the uncut versions of your two plasmids. Pipette 6.5 μl of TE buffer into each of the two tubes followed by 1.5 μl of 6X loading dye and 2 μl of your uncut plasmid DNA.
12. Complete Activity 3: DNA Model until your restriction digestions are ready.
13. Once your restriction digestions are complete, add 4 μl of 6X loading dye to each of your samples. Use separate tips for each sample to prevent cross contamination.

What is the role of “master mix” in restriction digestion? What does the term “digestion” mean in the context of this lab? What will be the final concentration of loading dye in each of your samples?

Activity 2: Agarose Gel Electrophoresis

In this activity, you will use prepoured agarose gels. The gels are made by weighing solid agarose powder, placing it into a flask, adding gel electrophoresis buffer, and boiling the mixture in a microwave oven until the agarose powder dissolves. When the gel solution cools to the touch, a DNA-detection reagent called SYBR Safe is added to the molten agarose. The agarose is poured into a comb-containing gel-mold, and allowed to cool and solidify. You will place the prepoured gel in an electrophoresis tank, add buffer, add your dye-treated DNA samples to wells in the gel, and apply voltage to separate your DNA fragments.

Learning Objectives

After successful completion of this activity, you should be able to:

• Label the sizes of standard bands in the DNA marker based on a key (LO112)
• Draw a DNA fingerprint of a plasmid DNA cut with a restriction enzyme for which the position of the sites is known (LO113)
• Match materials and equipment for agarose gel electrophoresis with their function (LO114)
• Diagram the relationship between mapping an unknown plasmid to show how to solve a case of an alleged “nosocomial infection” using DNA fingerprinting (LO118)

Running the Agarose Gel

Viewing the DNA

Activity 3: Exploring DNA with the DNA Discovery Kit©

In this activity your group will cooperate with the rest of the class to build a DNA model. You will also simulate the plectonemic nature of double-stranded DNA using toobers.

Learning Objectives

After successful completion of this activity, you should be able to:

• Label a picture or model of DNA of important chemical components and groups and bonds (LO109)
• Explain the different types of DNA in bacteria (plasmids vs. chromosomal (LO110)

Procedure

Nucleotides and Polynucleotides

Building a double-stranded DNA helix

Exploring the structure of double-stranded DNA

Data Analysis

Part A Complete this during the gel run.

1. Your instructor will give you a copy of the data analysis A worksheet. The worksheet contains a gel picture of the plasmid isolated from the defendant’s bacterial infection in uncut form and after digestion with both EcoRI and PvuII. The worksheet also contains a table of the size and location of EcoRI and PvuII restriction sites for the plasmids, A, B, C, and D isolated from bacterial infections from the four patients.
2. Estimate the size of the DNA fragments on the gel picture as follows, writing necessary information in your lab notebook:

Part B Complete this following the gel run.

11.2 Self-Assessment

These questions were taken from previous exams and are meant to represent a sample, not a complete study guide. The questions in these examples are designed to test your understanding of the concepts and skills presented in this lab, and your ability to apply what you have learned to novel problems.