Biology 100/101
Lecture 19
Biotechnology: DNA Analysis
(Print Version)

Announcements &
Assignments

Lecture Objectives

Web Resources

DNA Fingerprinting

DNA Sequencing

Gene Chips

Lecture Syllabus

IB 100/101 Home Page

Announcements
Text Readings
in Lewis, et. al.
DNA Fingerprinting
Chapter 12, Biotechnology 12.1, DNA Fingerprinting:
An Application of Understanding DNA Structure, pg 228-9

DNA Sequencing
Chapter 12, Biotechnology 12.2,
Two Routes to DNA Sequencing, pg 232-3

You may also ask questions and see answers to your classmates' questions in Web Crossing in the "Talk to Jim, Ross, and Ed" discussion.

Objectives

After studying this material you should be able to:

  1. Describe how gel electrophoresis is used to separate and order DNA fragments of various lengths.

  2. Explain the effects of mutation and inheritance of DNA that provide the theoretical basis for the use of DNA profile, also called DNA fingerprinting, as a tool for the identification of individuals or determination of the relationships of organisms.

  3. Describe the role of polymerase chain reaction (PCR) and gel electrophoresis in the preparation of a DNA profile.

  4. Briefly describe how DNA sequencing using "cycle sequencing" method is similar to and different from polymerase chain reaction (PCR) and DNA profiling.

  5. Define the term "microarray" AKA "DNA chip or gene chip" and describe how they are used as a biotechnology tool.
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Web resources:

Collection of Biotechnology Animations from Cold Spring Harbor
The animations dealing with PCR , Southern Blotting, and Cycle Sequencing are especialy good.

Cracking the Code of Life From NOVA

    This is another possibility for extra credit. Watch the entire program and write it up as a Media Watcher" extra credit project.

Gene chips or DNA arrays
Click on "Techniques" at the botom of the page and choose "Large-scale analysis" from the top of the next page.

DNA Fingerprinting (DNA Profiling)

DNA Fingerprinting, also called DNA Profiling, makes use of segments of DNA that do not code for protein products, but do exhibit variability (caused by mutation) in the nucleotide base sequences from individual to individual. Because the DNA is not involved in making important protein molecules, mutations have no affect on the health or survival of the individual. In some instances the segment of DNA being investigated contains varying numbers of repeated letters from one individual to another, like a molecular stutter. One segment of DNA on chromosome #5 that is routinely used in DNA profiling includes from 7 to 15 repeated sequences of the short sequence, AGAT.

DNA profiling is a highly standardized process. The FBI has set the standard and routinely uses 13 specific DNA locations on chromosomes 2, 3, 4, 5 (2 loci), 7, 8, 11, 12, 13 16, 18, and 21, in addition to the sex chromosomes, for DNA profiles for humans.

Each person inherits two versions of these variable length DNA segments on their chromosomes, one from her/his father and one from mother.

In DNA profiling, these segments of DNA are amplified using polymerase chain reaction (PCR), to produce 28 types of DNA fragments of various lengths that are unique to each individual. There is one type of fragment from each of the 13 loci from the paternal chromosomes and 13 from the maternal chromosomes, plus the two sex chromosomes.

Special single-stranded DNA primers that initiate DNA replication just upstream and downstream from the specific DNA fragments insure that only the desired segments of DNA are amplified. Each primer is about 25 nucleotides long. One of the two primers for each locus is "tagged" with a colored molecule that helps identify the DNA segment that it helps amplify.

After the DNA segments from a sample of DNA from a person are amplified and "tagged" with the colored primers they are "loaded" onto a gel electrophoresis apparatus and separated by length. An electric current passed through a porous gel causes the negatively charged DNA fragments to move through the gel toward the positive electrode. Smaller fragments move more rapidly. Longer fragments are impeded by the gel.

  • Electrophoresis animation from DNA Interactive.

      • Select "Techniques at the bottom of the screen
      Select "Sorting and Sequencing" from the top of the next screen.
      Select the "Gel Electrophoresis" animation.

    The DNA fragments are scanned by a laser probe as they pass through the electrophoresis gel. The colored primers attached to each fragment are detected as they pass the laser probe. Because each person has a different combination of DNA fragments of varying length, the pattern of colors detected by the laser probe is different for each person.

    See Lewis' Text: Biotechnology 12.1, DNA Fingerprinting: An Application of Understanding DNA Structure, pg 228-9

  • New Methods of DNA Profiling using PCR and STRs (Short Tandem Repeats). DNA Interactive: Applications
    • Select "Human Identification from the bottom of the first screen
    • Select "profiling" from the top of the second screen.
    • Roll your mouse over the right hand circle near the top of the screen ("Today's DNA Profile") and click to select the animation.

    Some Applications of DNA Fingerprinting (Some of these links feature an older method of DNA fingerprinting, but the basic concepts are the same):

    DNA Sequencing (figuring out the sequence of As, Cs, Gs, and Ts in a sample of DNA)

    DNA fingerprinting or profiling can be used to identify an individual based on his or her DNA, but a more precise form of DNA analysis is accomplished by actually determining the sequence of a particular segment of DNA.

    DNA Sequencing can be used to identify an individual, determine if an individual carries a particular mutation, or on a grander scale, determine the sequence of the entire human genome. See The Human Genome Project.

    One of two general techniques ("cycle sequencing" and DNA "chips") are used to determine the specific sequence of nucleotides in the sample to be analyzed. We will focus on the cycle sequencing method.

    Gene Chips - DNA Chips - Biochips - Microarrays - and how they work:

    Gene Chips or DNA arrays

      Click on "Techniques" at the botom of the page and choose "Large-scale analysis" from the top of the next page.

      The animation "DNA arrays" is a good summary of how gene chips are produced and used.

    Use of DNA Chips to study Autism from the BBC

    DNA Microarray Methodology from Davidson College

    Microarrays - (Yeast example)

    robot used to create microarrays

    A DNA microarray is a small glass slide upon which from a few to thousands of known samples of single stranded DNA are placed in very, very, very small dots.

    The type of DNA in each dot is mapped in a computer program

    Samples of DNA to be tested are labeled with a colored dye and applied to the known samples on the chip to see if any of the DNA in the sample matches up with and sticks to the DNA on the chip.

    Scanning the chip through a microscope and comparing the location of the colored dots with the computer map of the sample on the chip indicates which DNA is present in the sample.

    The chips may be used to:

    • Conduct genetic tests for the presence of a known mutation of a gene.

    • Determine which genes are active in a particular organ or in the presence of a particular environmental stimulus (hormone, light, nutrient, etc.).

    • Look for the presence of a particular organism or genetic material (testing for HIV genetic material, or testing a soil sample for a particular bacterial species.)

    • Sequence DNA

    • An Example using Microarrays to determine which genes are active in a tissue sample:
      • Samples of DNA from part of the genome or even each and every known gene sequence are applied to the surface of a glass slide in a series of microscopic dots - an array.

      • When a gene is activated it produces messenger RNA (mRNA) in the process of constructing the protein encoded in the gene.

      • Messenger RNA can be specifically extracted from cells in certain tissues and/or at different times of development.

      • The question to be answered is, "Which specific allele or mutated form of a gene or group of genes is/are present?"

      • The goal of microarray analysis is to match each type of mRNA extracted from a cell with the DNA from the gene that produced it.



      • An enzyme (reverse transcriptase) is used to produce complimentary DNA molecules (cDNA) using the mRNA extracted from the cells as templates.

      • The cDNA is labeled with a fluorescent dye.

      • The cDNA produced from the cell sample is unwound by heating and literally washed over the microarray of DNA samples on the chip.

      • Wherever DNA molecules from the sample match up with complimentary DNA molecules on the chip they bind.

      • The chip is then examined under a fluorescence microscope with laser light and a photograph is taken to record the fluorescence of dots on the chip that have hybridized with cDNA prepared from the cell sample.

      • The location of each and every segment of DNA on the chip is known, so determining which genes were active in the cell is just a matter of comparing the pattern of fluorescing dots to the gene map of the chip.