Biology 100/101
Lecture 19
Biotechnology: DNA Analysis
(Print Version)
Announcements
| Text Readings in Lewis, et. al. |
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DNA Fingerprinting Chapter 12, Biotechnology 12.1, DNA Fingerprinting: An Application of Understanding DNA Structure, pg 228-9
DNA Sequencing
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You may ask questions and see answers to your classmates' questions in Web Crossing in the "Talk to Steve and Ed" discussion.
Objectives
After studying this material you should be able to:
- Define the term Restriction Fragment Length Polymorphisms (RFLPs) and describe how they are separated by electrophoresis and identified using DNA probes.
- Explain the effects of mutation and inheritance of DNA that provide the theoretical basis for the use of DNA fingerprinting, also called DNA profiling, as a tool for the identification of individuals or determination of the relationships of organisms.
- Briefly describe how a segment of DNA is sequenced using the Sanger method and using DNA microarrays (DNA chips or gene chips).
- 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 and Southern Blotting
are especialy good.
Cracking the Code of Life From NOVA
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 individulat to individual. When these segments of DNA are cut using restriction enzymes DNA fragments of various lengths are produced. If the DNA of an individual has mutations within restriction sites the DNA will not be cut at those sites and that individual's DNA fragments will be different in length from another's DNA fragments produced by the same restriction enzymes.
When the DNA fragments of an individual are separated by electrophoresis, transferred to a nylon membrane using the Southern blotting technique, and mixed with specific radioactive single-stranded DNA probes, a unique pattern of bands is produced when the membrane is autoradiographed. These bands can be used to identify that individual.
See Lewis' Text: Biotechnology 12.1, DNA Fingerprinting: An Application of Understanding DNA Structure, pg 228-9
- PCR is used to amplify a particular section of DNA from a sample. The specific section can be from either the nuclear DNA or the mitochondrial DNA. The section chosen is usually NOT a functioning gene, but a highly variable, non-coding section of DNA.
- When the same section of DNA from a the same chromosome or mitochondrial DNA of different individuals is cut using the same restriction enzyme, fragments of DNA are produced called restriction fragments.
- RFLPs - Restriction Fragment Length Polymorphisms
- Samples of DNA from different individuals cut by the same type of restriction enzyme are separated by length using a technique called gel electrophoresis. The DNA samples that have been cut up by restriction enzymes are placed in small indentations in a sheet of porous material similar to gelatin. An electric current pulls the naturally, negatively charged DNA molecules toward a positive electrode. Shorter fragments of DNA get pulled farther through the porous gel than the longer pieces, separating the DNA into bands of different length.
- Probes probes are artificially produced, short sequences of DNA matching known segments of DNA (SImilar to the concept of DNA primers, used in PCR). They are labeled by radioactive or fluorescent atoms or molecules that allow the visualization of the probes once they have "stuck" to complementary sections of DNA - if they are present - in the DNA samples being tested or compared.
- Web Crossing Supplement - Electrophoresis and DNA Analysis
- Southern Blotting: Gel Transfer from Access Excellence (Similar to the Web Crosing Supplement above)
- DNA Fingerprinting Animation from Cold Spring Harbor Laboratory
Restrictriction Enzymes, RFLPs, Electrophoresis, and Probes: Here's the basic idea of how DNA fingerprinting works
Because each individual's DNA contains unique mutations inherited from their ancestors, the fragments of DNA produced by the same restriction enzymes (restriction fragments) may vary in length (be polymorphic - of different forms).
Some Applications of DNA Fingerprinting:
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.
PCR (again) is used to amplify a particular segment of DNA from a sample for study.
DNA Sequencing can be used to identify an individual, determine if aan 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 (the Sanger method, or DNA "chips" are used to determine the specific sequence of nucleotides in the sample to be analyzed.
- The Sanger Method of DNA Sequencing
- Lewis, Biotechnology 12.2, Two Routes to DNA Sequencing, (The Sanger Method) pgs 230-231.
- DNA Sequencing from Kimball's Biology Pages
- DNA Sequencing from the Cold Spring Harbor Biology Animation Library
- Sequence for Yourself from NOVA
- Lecture Activity: DNA Sequencing
Gene Chips - DNA Chips - Biochips - Microarrays - and how they work:
DNA Microarray Methodology from Davidson College
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 pprogram
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 onteh 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.).
- Microarrays - (Yeast example)
- 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.
