Danielle R Snowflack PhD wwwedvotekcom EDVOTEK The Biotechnology Education Company Celebrating OVER 30 years of science education Todays Experiment Restriction Enzymes allow researchers precisely cut DNA ID: 917266
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Slide1
Biotechnology Basics:
Restriction Digests using Kit 225
Danielle R.
Snowflack
, Ph.D.
www.edvotek.com
Slide2EDVOTEK
The
Biotechnology Education
Company
Celebrating OVER 30 years of science education!
Slide3Today’s Experiment
Restriction Enzymes allow researchers precisely cut DNA
Forensic scientists analyze DNA using restriction enzymes
Today’s Experiment: EdvoKit #225, DNA Fingerprinting using Restriction Enzymes
Slide4DNA sequences are unique
The chances of two individuals having exactly the same DNA profile is ~30,000 million to one
Single Nucleotide Polymorphisms (SNPs)
Restriction Fragment Length Polymorphisms (RFLPs)DNA profiling is a forensic technique used to identify individuals by analyzing differences within DNA.
CODIS – Combined DNA Index System
Slide5RFLPs are used as landmarks for
DNA Fingerprinting
Restriction Fragment Length Polymorphisms (or RFLPs)
are heritable differences in the nucleotide sequence.
Some RFLPS add a restriction enzyme cut site to the DNA.
Cat. # 109
A
B
Slide6Learn more about Forensic Science in our previous live streams
Cat. # 119
Forensic DNA Analysis
Forensic Blood Typing
Slide7Experimental Structure
Module I: Crime Scene Investigation - Restriction Enzyme Digestion
DNA samples digested by mixing with enzymes
Module II: Agarose Gel ElectrophoresisDNA fragments separated by size
Module III: Gel StainingDNA becomes visible
Slide8Samples: digested DNA
Agarose
Electrophoresis Buffer
Electrophoresis Apparatus
D.C. power sourceMicropipet
What do I need to perform electrophoresis?
Slide9Electrophoresis chambers for classrooms of all sizes
Cat. # 502
Model M12
Two gels
Cat. # 515
Model M36
Six gels
Slide10Power supplies provide current for electrophoresis
Cat. #5010
QuadraSource
™
(10-300V)Cat. #509
DuoSource
™
150
(75/150 V)
Slide11Introducing the Edvotek
EDGE™
Slide12Restriction digests
Restriction enzymes act like molecular scissors, cutting through the double-stranded DNA at specific, palindromic sequences.
Digestion of the same piece of DNA with different enzymes will create unique patterns.
https://
innovativegenomics.org/glossary/cleave/
Slide13The evolution of restriction enzymes
Restriction endonucleases evolved in bacteria as a defense against viral attacks.
If the enzyme recognizes foreign DNA, it chops the invader’s DNA into pieces to prevent replication.
Slide14Isolation of restriction enzymes
Identified and purified the DNA digestion activities from different bacterial species
The 1978 Nobel Prize honored “the discovery of restriction enzymes and their application to problems of molecular genetics."
Slide15Types of DNA ends
Type II enzymes are homodimers with DNA binding domains and endonuclease domains.
Many restriction enzymes recognize and cut palindromic stretches of DNA, generally 4-8 base pairs in length
Restriction fragments have “blunt” or “sticky” DNA ends.
Slide16How many times will DNA be cut?
The probability that an enzyme will cut DNA is proportional to the length of its recognition site.
Statistically, enzymes cut once every 4
n base pairs, where n is the length of the recognition site. Four BP site – one cut per 256 bases (4
4)Six BP site – one cut per 4096 bases (46)
Slide17Applications of restriction enzymes in the research laboratory
Molecular Cloning
allows researchers to isolate, combine, and reproduce specific DNA sequences
DNA Fingerprinting identifies individuals based on the sequence of their DNARestriction Enzyme Mapping describes the sequence of DNA by identifying the location of restriction enzymes.
Slide18Preparation of
Dryzymes
™
Edvotek™ kits provide restriction enzymes in a stable, freeze-dried form.
The Dryzymes™ arrive as a dry, flaky white powder which is resuspended in bufferLyophilization stabilizes perishable reagents, making it easier to ship and to store these materials.
Slide19Performing the restriction digest
Set up the experiment as per the experimental protocol.
Prepare and aliquot restriction enzymes and store on ice when not in use
For sections on multiple days, freeze aliquots at -20ºC. For best results, use within a week.For best results, the DNA must be digested at 37°C for 30-60 minutes.
Optional stopping point: After DNA digest, add loading dye to samples and then freeze until next lab section.
Slide20Summary of electrophoresis
Slide21Electrophoresis separates
DNA fragments by size
Samples are loaded into the wells, and an electrical current is passed through the gel.
The sugar-phosphate backbone of DNA has a strong negative charge.
The current drives the DNA through the gel towards the positive electrode.
Slide22Electrophoresis separates
DNA fragments by size
Small DNA fragments move through pores easily, but large DNA fragments have a more difficult time squeezing through the tunnels.
The DNA separates into distinct zones based on size.
Slide23Electrophoresis Separates DNA Fragments By Size
DNA is clear and colorless.
After the current is stopped, the bands can be visualized using a stain that sticks to DNA.
FlashBlueTM Stain
SybrSafe™ DNA Stain
Slide24DNA Stain
TruBlu
2 Transilluminator #557
SybrSafe® Stain #608
After the current is stopped, the bands can be visualized using
SybrSafe
, a stain that sticks to DNA.
Melt agarose and cool to 65°C.
Add concentrated SYBR® Safe stain to the molten gel at 1:10,000 dilution (5
μ
L per 50 mL agarose solution).
Run DNA samples through gel – no post staining or
destaining
necessary!
Slide25Considerations and Adaptations for Hybrid Learning
To save time, prepare electrophoresis gels in advance and store at 4ºC
If using
SybrSafe
in gel, store in a small bag with ~2 mL buffer.After electrophoresis, store gels at 4ºC.Store in a small bag with ~2 mL buffer.
Slide26Results and Conclusions
Each digested DNA will produce different fragment patterns.
A match provides strong evidence that the suspect was present at the crime scene.
Alone, this evidence does not prove the suspect committed the crime.
If a suspect’s DNA profile does not match that of the crime scene, that person may be eliminated from the inquiry.
Slide27Restriction Enzymes are an important part of the
Biotech
ToolboxRestriction Endonucleases evolved as an “immune system” for bacteria.
These powerful biotechnology tools cut DNA at specific nucleotide sequences.DNA Fingerprinting uses restriction enzymes to identify sequence differences in an individual’s DNA.
Slide28EDVOTEK, Inc.
The Biotechnology Education Company
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