DNA Laboratory Workshop

DNA Laboratory Workshop

SUNY Oneonta

Nancy J. Bachman, Ph.D.

July 10, 2007

Program sequence: (Session 1 10 am-12 noon; Session 2 1 pm-3 pm)

Classroom/laboratory, room 110 Physical Science

1. Introduction, laptop powerpoint slide show

2. DNA isolation from cheek cells.

3. PCR set up.

4. DNA testing gel

5. Sensors and molecular switches demos, room 111A Physical Science

Demonstration, room B18 Physical Science

6. DNA sequencing and forensic fragment analysis

Demonstration, room 35 Denison Hall

7. Fluorescence microscope and digital imaging

Cells and DNA

All living organisms are made of one or more cells. Different parts of cells have different functions, for example the nucleus contains the genetic material DNA and carries the blueprint for the cell.

DNA is short for deoxyribonucleic acid. The name refers to the type of sugar molecule contained in DNA, deoxyribose. In addition to the sugar group, phosphates and nitrogen-containing bases (adenine=A; guanine=G; thymine=T; cytosine=C) are the other parts of a basic subunit of DNA, called a nucleotide.

DNA consists of a long chain (polymer) of nucleotides. The order of bases determines the genes which specify the traits of a person (eye color, dimples, etc.).

Many techniques in biotechnology and DNA nanotechnology depend on the complementary pairs of bases in DNA. The A bases (adenines) always pair with T bases (thymines) and the G bases (guanines) always pair with C bases (cytosines). New DNA is made from old DNA (this is called replication) following these rules. DNA can “unzip” to expose a strand and these can be copied down the line to make a new strand. DNA synthesis is the basis for PCR and DNA sequencing methods you’ll hear more about today. Also the complementary pairing of strands enables two strands to find each other and “zip up”; this idea is the basis of DNA tiling and DNA computing technologies.

DNA Extraction from Cheek Cells

Distribute to participants before start:

• Paper cup
• Sterile swab
• Numbered microcentrifuge tube with 0.5 ml quick extract DNA solution

Prepare before start

• Water bath at 65°C
• Heat block at 98°C
• Vortex mixers

1. Take a paper cup to the drinking fountain and rinse your mouth twice with clean water.

2. Take a swab and firmly brush the inside of your cheek 20 times on each side.

3. Place the swab end into the microcentrifuge tube and rotate in the solution at least 5 times.

4. Mix on vortex for 10 seconds.

5. Incubate the tube at 65ºC for 1 min.

6. Mix on vortex for 15 seconds.

7. Incubate the tube at 98°C for 2 min.

8. Vortex tube for 15 seconds.

9. This tube now contains your DNA!

Background on PCR: Polymerase Chain Reaction

Very small amounts of biological samples (a single human hair, a cheek cell, or a drop of blood) can provide enough DNA (deoxyribonucleic acid, the hereditary material) for tests to help identify victims of crimes and to match criminal suspects to evidence.

A method used to reproduce the small amount of DNA to make many more copies of it is called PCR, Polymerase Chain Reaction. This procedure uses the same raw materials a cell uses to reproduce DNA--a small amount of DNA isolated from the evidence, "primers" (short bits of DNA that start the new strands), nucleotides (the subunits that make up DNA), and the enzyme that synthesizes DNA (DNA polymerase). The reaction is set up in a small plastic tube (holds 0.2 ml) and is carried out in an instrument called a thermocycler, programmed to run a series of steps under different temperature conditions.

Before you set up the reactions, first practice using a micropipet. This is a device designed to measure very small liquid samples (usually from about 1 to 1000 microliters; remember a liter is about a quart and a microliter is 1/1,000,000 of a liter). A staff member will demonstrate and give you samples of water to practice with. A disposable tip (these have filters to prevent contamination of samples) is placed on the end. The plunger is pressed down just until you meet resistance and the pipet tip is inserted into the liquid. The liquid is then drawn up and transferred to the reaction tube. This time you press the plunger nearly all the way down to expel it. Discard used tips in the container provided.

PCR from human DNA

1. You will be given a tube with all of the components of the reaction, except the DNA sample (total of 23 microliters). This is called the “Master Mix” and it contains nucleotides, primers, buffer, magnesium ion and Taq DNA polymerase, along with dyes that make it green.

2. Label your Master Mix tube with the number code for your sample with a Sharpie pen.

3. Using a micropipet and a disposable filter tip, transfer 2 microliters of DNA into the tube.

4. Close the tube and return it to the rack.

5. The reactions will be covered with a drop of oil and we will demonstrate the BioRad “MyCycler” thermocycler. The instrument runs through three temperature steps in a "cycle".

• Heating to 94˚C separates the strands of DNA
• Cooling to 45-60˚C allows the primers to bind the strands of DNA
• Heating to 72˚C enables the enzyme to synthesize DNA

We will program the machine to run 30 cycles. Assuming optimal conditions, this means there will be 230 copies of the original DNA after 30 cycles!

Background: DNA Fingerprinting

Sources:

1.

2.

DNA Fingerprinting is a powerful new technology that has dramatically changed forensic science and has become popularized in television crime shows, such as CSI. DNA fingerprints are unique to each individual (except identical twins) and can be used to establish family relationships and to determine whether evidence left at the scene of a crime comes from a particular suspect. This information is especially useful for solving sexual assault and murder cases.

paternity casescriminal cases

What does DNA fingerprinting involve?

Most humans have very similar DNA patterns. However, sometimes when DNA is copied, mistakes can be introduced in the DNA; these are known as mutations. Mutations make each individual persons’ DNA sequence unique.

DNA fingerprinting can identify the different forms of DNA in each individual. Most of the techniques used in forensics detect individual differences in the number of copies of a short sequence repeat (such as AGAGAG or CAGCAGCAG), often referred to as VNTRs (variable number of tandem repeats).

More copies of a repeat make the DNA strand longer. Differences in DNA length can be detected by a method called gel electrophoresis.

In gel electrophoresis, molecules of different sizes can be separated in an electric field. The molecules of DNA are negatively charged (due to the presence of phosphate groups) and are attracted to the positive pole (opposite charges attract). They travel through the pores of the gel at different rates; short DNA molecules travel faster and farther than long DNA molecules.

Using DNA fingerprinting in criminal cases

A single DNA fingerprinting test looks for variation at a particular location (locus) on the DNA. Depending on the test, many individuals may have the same test result or few may have the same test result. Usually the results from a number of DNA tests are used to establish innocence or guilt in a court of law. For example, if four different tests are run, the likelihood of identifying an individual with exactly the same pattern is compounded as each additional test result is added. In the example below, the odds of identifying the correct individual increase from 1 in 25 if just the first test is done to 1 in over 60 million, if all four tests are done.

Locus tested / Frequency in population / Combined frequency
1 / 1 in 25 / --
2 / 1 in 100 / 1 in 2500
3 / 1 in 320 / 1 in 806,000
4 / 1 in 75 / 1 in 60,600,000

Reference: Cummings, M. (2003) Human Heredity, Principles and Issues. Brooks/Cole, Pacific Grove, CA. p. 325.

DNA Fingerprinting Gel Demo

1. The gel should be submerged in 1x TBE (electrophoresis buffer). Make sure the tray is oriented so that the wells are located closer to the black (negative) pole and away from the red (positive) pole.

2. Loading DNA samples:

a. Practice loading samples. DNA samples are typically mixed with loading dyes and inserted into wells. You will practice loading 10 µl of diluted loading buffer into a sample well (Fig. 1).

Fig. 1. Loading the gel

b. The demo gel you will see represents a DNA fingerprinting case. Ten microliters of each sample were loaded into the appropriate gel lane.

Lanesample

1 / Size markers
2 / Suspect 1
3 / Suspect 2
4 / Suspect 3
5 / Suspect 4
6 / Suspect 5
7 / Suspect 6
8 / Suspect 7
9 / Suspect 8
10 / Suspect 9
11 / Suspect 10
12 / Suspect 11
13 / Suspect 12
14 / Suspect 13
15 / Suspect 14
16 / Evidence

2. The instructor connected the cover and the electrodes so that the DNA samples migrated toward the positive (red) pole. REMEMBER: RUN TO RED! A typical small gel takes about 1 hour to run.

3. We will view the gel results in a darkened room. A fluorescent dye, ethidium bromide, binds the DNA and glows bright pink on a UV (ultraviolet) light source.

Sensors and Molecular Switches

“Biocomputing” uses components of cells, such as DNA, proteins, and membranes to carry out calculations, to serve as the on/off switches that tell a computer what to do, or to signal to us what the result is. In this demonstration, you will see that molecules can cause color changes (such as white to blue), light emission (luminescence), and fluorescence (when a molecule absorbs at one energy and emits at another energy). We will also demonstrate how some of these molecular indicators are helpful in forensics.

THE LACTOSE OPERON: A molecular switch.

The Lactose Operon is a group of genes in the E. coli bacterium that determines whether or not it can use and break down the sugar, lactose (milk sugar). An operon is a molecular switch; in this case the genes are mostly switched off unless lactose is around. Scientists (Jacob and Monod) learned how the switch worked by finding out what happens when the switch is broken.

In normal (often called “wild type”) bacteria, the switch is off when lactose is absent; but the switch is on when lactose is present. We can tell the switch is on if we put in an indicator (called X-gal) for short, which turns blue when the gene for the enzyme β-galactosidase is turned on. When the operon is off, little or no β-galactosidase is produced (color is pale blue).

When certain components of the operon are broken (such as by a mutation in certain regions of the DNA), the switch may be off all the time (color is white). This can happen if either the promoter (lacP), a regulatory element, or the β-galactosidase (lacZ) gene is broken:

White streaks are lac Z- (mutant) E. coli; blue streaks are lacZ+.

Molecular Light Switches

Firefly

Many organisms such as fireflies can make light from a simple chemical reaction. These organisms usually have the gene for an enzyme called “luciferase”. Luciferase allows them to “glow in the dark”! We will demonstrate the presence of a luciferase gene (actually not from a firefly, but from a bioluminescent bacterium called Vibrio), inserted into E. coli. Be patient as your eyes adjust to the dark room for the demonstration.

View of plate in the light vs. view in the dark (bioluminescence)

Chemical reactions can also produce light; this is the basis of the luminol test used in forensics. Luminol reacts with the iron in blood hemoglobin (the protein in blood that carries oxygen) in a chemical reaction that produces a greenish-blue glowing light. Even trace amounts of blood left after a hasty clean-up can be detected.

Luminol test for blood stains

Luminol test

1. Perform in designated darkened area or room. Place the paper or fabric with possible blood stains on a piece of absorbent paper.

2. Spray with the luminol reagent (previously made by mixing 1 vial of Evident luminol with 4 oz. water in a small spray bottle).

3. Look for the immediate blue-white chemiluminescent reaction.

FLUORESCENCE

Green (and Blue and Yellow and Red and Cyan) Fluorescent Protein

Fluorescence occurs when a molecule absorbs light at one wavelength, and emits light of a longer wavelength. It can be used as a molecular sensor and has been extremely useful for tracking molecular events in cells. Many small molecules fluorescence; also extremely useful has been the discovery of Green Fluorescent Protein (GFP)in the jellyfish, A. victoria. You will see a demonstration of GFP inserted into E. coli under control of an artificial “molecular switch” that enables the fluorescence to be switched off.

GFP is also used to create transgenic organisms, such as this “green bunny”.

Fingerprinting with fluorescent dyes

Fingerprints are formed by sweat. When a finger touches a surface, the sweat from pores is deposited, providing a mirror image of the ridge pattern. The shapes of the ridges can be described in terms of arches, loops and whorls. No two persons and no two fingers have the same ridge pattern and patterns do not change with time, making each fingerprint unique. Traditional fingerprint powders were black or white; new types of fingerprinting powders are magnetic or fluorescent or a mixture of the two.

Fingerprint ridge patterns

Fluorescent powder (orange or green)

1. Use the orange feather duster for the orange fluorescent powder and the green feather duster for the green fluorescent powder.

2. Dust the fluorescent powder on and off the print using the feather duster.

3. Use a black light in a designated dark room to see the fluorescence.

Magnetic fluorescent powders (orange or green)

1. Use the magnetic wand to transfer the powder to the print.

2. Use the magnetic wand to pick up the excess powder and transfer back to stock container. Use a tissue to remove the remainder of the powder from the wand.

3. Use a black light in a designated dark room to see the fluorescence.

DNA sequencing: Reading the Bases on DNA

In 2000, a major announcement was made that the entire human genome sequence had been determined. This would enable us to determine the code for all the genes and to study further the structure and regulation of genes (how they switch on and off). You will see a demonstration of the kind of automated DNA sequencing system that made it possible to determine the order of all the bases on DNA (A, C, G and T bases). Several companies make automated systems, these differ in the chemistries they support, the dyes that are detected and the way in which the electrophoresis (separation) is carried out.

Beckman automated sequencing systemCapillary electrophoresis separates DNAs

To carry out DNA sequencing, the DNA is first purified, then new DNA is synthesized in the presence of dyes to create chains of different lengths, using a thermocycler. The strands with dyes attached are separated by electrophoresis and pass a laser detector. The raw data from the instrument is converted to a DNA sequencing “trace”, a series of colored peaks that represent the order of the bases on a region of DNA.

From the trace the DNA sequence can be determined in the 5’ to 3’ direction:

5’______3’

Fluorescence Microscopy: Seeing the World of the Small

Fluorescence is when a molecule absorbs light at one wavelength and emits light of a longer wavelength. Cell biologists use fluorescent dyes to visualize different cell components and to investigate molecules in a cell and test their functions.

Microscopes use lenses to magnify small objects. A variety of microscopes are used in Cell Biology, Physics, Earth Sciences and Engineering applications to examine materials and living things. You will see a demonstration of an epifluorescence research microscope and digital imaging workstation, used to study cell structure and function. Here is a diagram showing how the microscope works:

A special lamp (here producing blue light) is shined on the specimen; if fluorescent, it absorbs the light and emits fluorescent light of a different wavelength (here, shown in green). You will also see some examples today of fluorescent images produced from a more sophisticated microscope (a confocal microscope). In a confocal microscope, the excitation light is provided by lasers; three dimensions of an object can be reconstructed because the microscope can focus on successive planes up and down (z-dimension) and the computer can reassemble these in a three-dimensional form.

1