Intro to Gel Electrophoresis & Restriction Enzyme Cleavage Patterns of DNA Part 1 & 2

recognition sites for restriction enzymes are usually

*4 to 8 base pairs

Practice Gel Loading

*After gel solidifies, place under DI water in a weigh boat. *Agarose gel nicknamed "submarine gel" bc samples are loaded under water, meaning buffer. *Practice delivering 40 microliters of gel loading solution to sample wells.

Preparing Gel for Electrophoresis

*After the Gels have solidified, slowly remove the dams from the gel bed. *Remove the combs by pulling straight up. *Place gel on its bed into the electrophoresis chamber, centered and level on the platform *Make sure gel is completely cover with buffer.

Restriction Enzymes

*Cut DNA *Isolate a gene *Made from bacteria *Recognize palindromes

Chamber Buffer Solution

*Ea. chamber requires 1 liter of diluted buffer. *2 liters are being used to run two gel labs. *mix 10 mL of buffer concentrate to 490 mL. *Mixed twice per chamber for 1 L of diluted buffer.

Restriction Enzymes

*Enzyme that cuts DNA at a specific sequence of nucleotides *Originally isolated from bacteria *Used for molecular cloning and DNA sequencing experiments.

10. What conclusions are drawn from analysis of results in the second gel experiment for Sample A? Sample B? Sample C? Sample D? Sample E? Sample F?

*Lane 1 is supposed to show three stained bands of uncut plasmid DNA that takes three forms: supercoiled, nicked, and a dimer form. The supercoiled form, being most linear in shape, migrates the furthest in lane 1. The nicked form migrates a little less, the idea being that a nicked plasmid completely unravels to a circular form. The dimer, or catenane form migrates the least, bc it represents plasmid replication. During replication, plasmids form interlocking circles, called dimers for two, trimers for three, and collectively called catenanes. Catenanes migrate more slowly than single circles that are nicked. *Lane 2 results one stained band, as the plasmid DNA has one recognition site for BgII. *Lane 3 show two stained bands, as plasmid DNA has two recognition sites for EcoRI. *Lane 4 have only one stained band, and it should not migrate very far at all. It was lambda DNA, uncut. *Lane 5 should show multiple fragments, probably 5 of them, bc lambda DNA contains five recognition sites for EcoRI. *Lane 6 will show so many fragments it will look almost like a smear. Lambda DNA has 29 recognition sites for BgII. When DNA fragments are of similar size, they run together in the gel.

Supercoiled DNA

*becomes linear in shaper *if appears in a nicked form, the phosphate bong was cleaved anywhere in the molecule, in either strand (Causes a twisted compact form like supercoiled to unravel to a circular form.)

Plasmid DNA Shape

*circular, and one form is supercoiled, which is like a twisted rubber band. *There are non-supercoiled such as linear, nicked, and relaxed circles. *approximately 4,500 base pairs

Catenanes

*interlocked circular molecules following replication. *they contain 2 or 3 plasmid molecules, & migrate slowly through a gel than a single circle.

Restriction Enzymes are labeled with a specific naming pattern:

*labeled as 1) the first letter of the genus 2) first two letters of the species 3) the type of strain or substrain 4) Roman numeral that denotes one out of several restriction enzymes produced by the same enzyme

Lambda DNA

*marker DNA, famous virus and helps to know the size while doing DNA fingerprinting *DNA already cut with two restriction enzymes *isolated from a bacteriophage *linear & contains 49,000 base pairs

Agarose Gel Electrophoresis

-is a separation method that is used to observe DNA segments or fragments that have been cut at specific sites by restriction enzymes. -It can be used to approximate size of DNA fragments in the range from 500-300,000 base pairs.

Apparatus (Chamber)

-is an horizontal gel electrophoresis apparatus. - contains electrodes at each end, & uses a gel made from agarose, a polysaccharide.

Second Gel Run Contains six DNA samples

-three are called plasmid DNA -three are called lambda DNA

4,500 base pairs

1) Circular --> Third Quickest 2) Linear --> Second Quickest 3) Supercoiled --> Travels the Furthest (First Quickest)

Prokaryotes when cut

1) It's circular, and it's cut once 2) Now it's a line (linear chromosome) and it's cut once into 2 lines 3) Prokaryote has now been cut twice. *Size will not affect how fast it moves *Shape moves faster - line moves quicker

Eukaryotes when cut

1) Linear before it's cut 2) Cut once = 2 Fragments traveling at the same speed (2 bands)

Gel Electrophoresis Steps

1) Make the gel 2) Set up gel apparatus 3) Load DNA sample into gel 4) Hook up electric current and run gel 5) Stain gel and analyze results

Batch Gel Prep

375 ml of 0.8% agarose gel *Take 500 or 600 mL beaker *Add 7.5 mL of buffer concentrate *Add 367.5 mL of DI water *Add 3 g of UltraSpec-Agarose; swirl to disperse clumps. *Heat agarose solution to boiling *Cool to 55degreesCelsius. Dispense 30 mL of cooled agarose solution to 7 x7 casting gel and allow to solidify.

Lambda DNA has

5 recognition sites for EcoR I and 29 for BgI I.

Variable base-position

5'-C Py CG Pu G-3' 3'-G Pu GC Py C-5'

1. On what basis does agarose gel electrophoresis separate molecules?

Agarose separates molecules based on its charge, size and shape.

BgI I was named for which organism?

Bacillus globigii.

Distilled Water

Cannot be used because it contains no ions, or electrolytes (charged particles in solution).

Linear DNA

DNA in Eukaryotes; restriction enzymes made based on gene it makes.

Palindromes

DNA sequence characteristic of restriction enzyme recognition site Frequently symmetrical; both DNA strands in the site have the same sequence when read 5' to 3'.

The second gel uses two:

EcoRI and BgI, I.

EcoR I was named for which organism?

Escherichia coli, strain RY 13

7. Why has the discovery of restriction enzymes been so important? How does this relate to their function?

Important because they allow the cleavage of DNA at specific sites; they are a tool for molecular cloning and DNA-sequencing methods.

Glycerol in gel electrophoresis

Increases the density of the samples, allow the sample to sink to the bottom of the well, instead of dispersing.

For the second gel, the order is:

Lane - Label - Sample - Cutting *Lane 1 - A - Plasmid DNA, uncut - circular *Lane 2 - B - Plasmid, cut with BgII - line *Lane 3 - C - Plasmid, cute with EcoRI - 2 Fragments *Lane 4 - D - Lambda DNA, uncut - line; single band; not very far *Lane 5 - E - Lambda DNA, cut with EcoRI - cut 5 times; 6 fragments *Lane 6 - F - Lambda DNA cut with BgII - 29 Recognition Sites, cut 29 times; 30 fragments

9. How are restriction enzymes named?

Names consist of the first letter of genus, first two letter of species, type of strain or substrain may follow as a capital letter; Roman numerals designate one out of possibly several restriction enzymes produced by the same organism.

6. What would happen if distilled water were substituted for buffer in either the chamber solution or the gel solution?

Nothing; won't work.

Bacteria

One Source of DNA Plasmid (4,500 bp long) Circular

2. Explain migration according to charge. To which electrode does DNA migrate and why?

Opposites attract.

Blunt End:

Restriction fragments with no overlapping ends resulting from cleavage by a restriction enzyme 5'-GG CC-3' 3'-CCGG-5'

Bacteriophage

Second source of DNA (49,000 bp) lambda DNA

4,500 circular base pairs

Single Circle - Faster due to shape affect it Two Circles - Slower & Bigger - called a Dimer Three Circles - Trimer Four Circles - Tetramer

Shape

The more linear the overall shape, the faster the sample migrates through the gel. It's important in the case of DNA, but not a factor of organic dyes.

4. What conclusion can be drawn from the results of the remaining samples in the first gel experiment?

The remaining samples all had net negative charges. The farther the migration rate in the lane, the smaller the sample was.

8. What are restriction enzymes?

They are endonucleases. They recognize and cleave DNA at specific sites known as palindromes. They were isolated from bacteria. In lab, they are used as tools for cloning.

InstaStain Methylene Blue

This step is used in second gel ONLY.

5. Why is glycerol added to the solutions before they are loaded into the wells?

To add density to sample to make it sink to the bottom of the well.

Charged Molecules

Travel in an electrical field based on net charge and mass.

After power supply is turned on

any charged molecules in the sample will migrate towards the electrode of opposite charge.

Charged Particles

are needed to conduct electricity

Fragments that are closest

are positive.

Fragment that end up at the end

are the furthest and most negative (toward (+) electrode).

DNA fragments move through gel toward positive electrode

begin at cathode (-) and travel to anode (+)

Two gel casting trays

called gel beds 7 x 7 cm each

Charged Dyes or Dye Mixtures

can be separated by agarose gel electrophoresis

gel electrophoresis process

enzymes are used to chop DNA from sample into fragments, fragments are then placed into wells aligned along the center of gel in the electrophoresis machine; electrical current is run through the gel and fragments are attracted to either side depending on charge; biggest/heaviest/least charged do not move far, smallest/lightest/most charged move far, thus creating banding pattern used to identify and compare

One quickstrip sample

filled with colored dye.

The objective of second run is to

identify restriction endonuclease, and understand how they are used as tools to cut DNA at specific sequence.

Color

is not a factor in mobility.

Diluted Buffer

maintains conductivity of the fluid; this allows the molecules to migrate in the gel.

Two blue casting combs

one placed in the second or middle set of notches in a gel bed for first experiment, and second one placed in the first set of notches in a gel bed in second experiment. Each comb has 6 teeth.

Plasmid DNA has

one recognition site for BgI I and two for Eco R I.

Charge

opposites attract

3. What conclusion can be drawn from the results of sample F, in the first gel experiment?

sample F is the only one with a net positive charge, thus migrated towards negative electrode.

Circular DNA

the DNA of prokaryotes; Entire DNA genome inside bacteria.

The apparatus takes advantage of

the negatively charged phosphate in DNA nucleotides to allow them to migrate towards the positive electron (red).

Size

the smaller the sample size (everything equal), the faster the migration rate, & the farther the sample will migrate through gel.

The most negative particles

travel the farthest.

net negative charge

travel to the positive electrode (the anode)

net positive charge

travels to the negative electrode (the cathode)

Charged DNA samples at a neutral pH

will migrate on the basis of charge, size and shape.