Analysis of DNA Fragments with Gel Electrophoresis
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Transcript of Analysis of DNA Fragments with Gel Electrophoresis
PRE-AP BIOLOGY
Analysis of DNA Fragments with Gel Electrophoresis
Bellwork
Explain how DNA fragments are created.
Objectives
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Define electrophoresisExplain the relationship between agarose
concentration and pore spaceExplain the role of the buffer in electrophoresisExplain how DNA is detected in agarose gels
Key Terminology
AgaroseElectrophoresisPore SpacesCombLoading Wells
Overview of Gel Electrophoresis
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Electrophoresis Uses electricity to separate molecules Used to analyze biological molecules Uses a gel to separate molecules based on charge,
shape and/or size
Biological molecules most commonly analyzed by gel electrophoresis include DNA RNA Proteins
Two Types of Gel Electrophoresis
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Classified based on orientation of gels Horizontal
gel lays flat commonly used for separating DNA fragments
Vertical gel is upright commonly used for separating proteins
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http://www.bio-link.org/vlab/Graphics/Tools/DNAGel.jpg&imgrefurl
Gel commonly used to analyze DNA
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Agarose (carbohydrate derived from seaweed) Separates DNA fragments ranging in size between 500
and 25,000 base pairs (bp) Separates DNA fragments by acting as a molecular
strainer Smaller fragments move through the pores easier than
large ones
Pore Size created in Agarose
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The number of hydrogen bonds between and within the carbohydrate chains of agarose create the pore size
The greater number of hydrogen bonds the smaller the pore size
Pore Size Analogous to Textile Weave
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g
Loose Weave Tight Weave
• Loose weave (fewer hydrogen bonds)•larger spaces between fibers
• Tight weave (more hydrogen bonds)•smaller spaces between fibers
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TThinkhink-P-Pairair-S-SharehareWhat is the purpose of the
agarose gel?
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Agarose Gel Preparation:
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Dissolve powdered agarose into electrophoresis buffer
Boil to dissolve the agarosePour into gel trays and insert comb to form loading
wells Allow to harden at room temperatureRemove comb to reveal loading wellshttp://
www.nslc.wustl.edu/courses/Bio2960/labs/07DNA/Gel/f21.gif
Electrophoresis Apparatus
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Place solidified gel in a gel box and cover with electrophoresis buffer
Electrophoresis Buffer commonly used is TAE Buffer pH 8
Purpose of the buffer Stabilizes the pH Maintains shape of DNA molecule Conducts electricity
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Electrophoresis Apparatus
The gel box has electrodes at each end of the box
anode (negative electrode, black) cathode (positive electrode, red)
The power supply that generates the electrical field is attached to the electrodes of the gel box
Movement of DNA in an Electric Field
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DNA molecules are negatively charged because of the phosphate
groupsUnder an electric field
they will move towards the positive electrode (cathode)
The smaller DNA fragments will move farther down the gel because they more easily move through the pore
spaces
TThinkhink-P-Pairair-S-SharehareExplain how
electrophoresis works IYOW.
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Detection of DNA in Agarose Gels
DNA can be detected by using a stain: Methylene Blue Works by bonding with the DNA and turning it dark
blue
Put the steps outlined on the Put the steps outlined on the next slide in ordernext slide in order
Be prepared to shareBe prepared to share
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Steps of gel electrophoresis for analyzing DNA fragments
Pour agarose gelIsolate DNAInsert electrodes into power supplyLoad agarose gel with DNA fragmentsCover agarose gel with electrophoresis bufferLoad DNA samples in agarose gelStain gel with methylene blueCut DNA with restriction fragments
DNA Fingerprints
DNA Fingerprints Way of identifying individuals By analyzing sections of DNA that vary widely
between individuals DNA fingerprints are created by
cutting the DNA with restriction enzymes separating those fragments on agarose gels
DNA Fingerprints important for
Forensics for identifying who is responsible for a crime
Paternity/maternity suits for identifying who is the father/mother of a child
Identification of bodies, for example after 9/11
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2 Minute Paper2 Minute Paper
You have two minutes to write down how agarose gel electrophoresis would be useful in identifying an individual’s DNA fingerprint.
Be prepared to share.