PRE-AP BIOLOGY 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

http://www.connecticutvalleybiological.com/http://www.connecticutvalleybiological.com/images/bt500.jpg&imgrefurlimages/bt500.jpg&imgrefurl

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.