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Hello you. Try this.Hello you. Try this.
2-1-17 If an enzyme was able to cut this DNA at each “x”, how many fragments of DNA would be produced? What fragment would be largest? Smallest?
x
x x xx
Your goal – to get get the basic idea of the basic idea of each lab each lab – what we will do for each
Today’s Goal
AP Biology AP Biology 1.1.Restriction Digest and Restriction Digest and
Analysis of Analysis of λλ DNA DNA2.2. Bacterial TransformationBacterial Transformation3.3.DNA Fingerprinting/Crime DNA Fingerprinting/Crime
Scene Analysis Scene Analysis
1. Restriction Digest of Lambda DNA1. Cut the DNA from a virus with enzymes and
analyze the results2. Bacterial Transformation
1. Cut out a gene from the DNA of one organism and insert it into the DNA of another
3. DNA fingerprinting1. Cut several DNA samples with enzymes and
analyze them to look for similarities & differences
Our 3 labs
Restriction Digest & Analysis of Lambda DNA
Examine restriction enzymes and Examine restriction enzymes and their use as biotech toolstheir use as biotech tools
Goal of this lab
useuse restriction enzymes EcoR1, Pst1, & HindIIIEcoR1, Pst1, & HindIII to digest (cut) bacteriophage lambda DNA
use gel electrophoresis to examine these fragments of DNA
What we will doWhat we will do
These enzymes cut DNA at very specific These enzymes cut DNA at very specific locationslocations Bacteria contain these enzymes as a Bacteria contain these enzymes as a defense mechanism against phagesdefense mechanism against phages Restriction enzymes are named after the bacteria from which they were first isolated • EcoRI from Escherichia coli, strain RI• HinDIII from Haemophilus influenzae, strain DIII• PstI from Providencia stuartii, stain I
What are restriction restriction enzymesenzymes?
• The DNA of a bacteriophage • ~ 48,000 base pairs long• 3 different enzymes to cut this DNA
resulting in different length restriction fragments
•one sample of DNA uncut for comparison•One “ladder” or “marker” used for estimating sizes of restriction fragments using gel electrophoresis
Lambda DNALambda DNA((λλ) )
If a linear strand of DNA is cut 4 times, how many bands will there be in our gel?
Restriction Enzymes
Electrophoresis - to carry w/ electricityElectrophoresis - to carry w/ electricitySeparates DNA fragments by sizeSeparates DNA fragments by sizeDNA fragments loaded into an agarose gel slab• Slab placed in chamber w/ conductive buffer• Direct current passed through• Since DNA molecules are negatively charged, they are Since DNA molecules are negatively charged, they are drawn toward the positive pole (anode) when placed drawn toward the positive pole (anode) when placed in an electric fieldin an electric field
The gel acts like a sieveThe gel acts like a sieve through which smaller fragments move more easily than larger ones Over time, smaller fragments will travel farther than larger ones
Agarose Gel Electrophoresis
Gel Electrophoresis
http://learn.genetics.utah.edu/content/labs/gel/
DNA is colorless, so a loading dye is DNA is colorless, so a loading dye is added to the DNA solutionadded to the DNA solution•Does not stain the DNA•Makes it easier to load the gels and monitor the progress of the process
Each restriction enzyme Each restriction enzyme produces unique banding produces unique banding patternspatternsThe relative size of the size of the fragments can be determinedfragments can be determined by measuring how far each band traveled from its origin
What are we doing overall in this lab?
So…
pGLO Bacterial Transformation Lab
Bacterial Transformation
DNA splicing, or gene splicing, is the cutting and DNA splicing, or gene splicing, is the cutting and linking of DNA molecules and it is one of the basic linking of DNA molecules and it is one of the basic tools of modern biotechnologytools of modern biotechnologyThe basic concept is to remove a functional DNA fragment, like a gene, from one organism and combine it with the DNA of another organism in order to study how the gene works• The desired result is for the recipient organism to The desired result is for the recipient organism to express the newly acquired geneexpress the newly acquired gene
Background
The ability to cut & paste, or cleave & ligate, a functional piece of DNA is what enables scientists to recombine DNA moleculesFirst step is to locate the geneNext use a restriction enzyme to use a restriction enzyme to cut out the gene from the rest of cut out the gene from the rest of the chromosomethe chromosomeUse the same enzyme to cut open same enzyme to cut open the DNA of the recipient DNA and the DNA of the recipient DNA and then insert the fragmentthen insert the fragment
Recombinant DNA technology
In this lab we will perform genetic transformation•Genetic transformation occurs when a cell takes up and expresses a new gene
We will learn about moving DNA from one organism to another using a plasmidplasmid
Overview GFP
Beta-lactamase
Ampicillin Resistance
pGLO™ & GFP
We will attempt to have our bacteria express the gene for green florescent protein (GFP)
GFP is a visual markervisual marker
Study of biological processes (example: synthesis of proteins)
Localization and regulation of gene expression
Cell movement
Cell fate during development
Formation of different organs
Screenable marker to identify transgenic organisms
Links to Links to Real-Real-worldworld
In addition to their single circular chromosome, bacteria often have bacteria often have 1 or more small, circular pieces of 1 or more small, circular pieces of DNA called plasmidsDNA called plasmids5- 6 genes in a circlePlasmids often contain genes that help bacteria surviveBacteria can share these plasmids with each other• Antibiotic resistance among bacteria is due to plasmid sharing
What is a plasmidWhat is a plasmid?
Transmission electron micrograph
Contains the Contains the genes for GFP genes for GFP and the gene and the gene for for ampicillin (antibiotic) resistanceampicillin (antibiotic) resistance• It is called the pGLO plasmidOur plasmid also has an operon that allows the gene for GFP to be turned on and offthe sugar arabinose turns on the operon
Our plasmidOur plasmid
Beta Lactamase•Ampicillin resistance
Green Fluorescent Protein (GFP)•Aequorea victoria jellyfish gene
araC regulator protein•Regulates GFP transcription
operon
Green fluorescent protein
Gene to break down antibiotic
Origin of replication
Bacterial Transformation
Beta lactamase(ampicillin resistance)
pGLO plasmids
Bacterial chromosomal DNA
Cell wall
GFP
Transformation Procedure
What are we doing overall in this lab?
So…
DNA fingerprinting
• Crime scene
• Human relatedness
• Paternity
• Animal relatedness
Anthropology studies
Disease-causing organisms
Food identification
Human remains
Monitoring transplants
DNA Fingerprinting Real WorldApplications
DNA Restriction Enzymes • Evolved by Evolved by bacteria to bacteria to protect against protect against viral DNA viral DNA infectioninfection
•Endonucleases = cleave within DNA strands
•Over 3,000 known enzymes
No 2 people are exactly the same genetically except…• Identical siblingsAll people share 99.9% same DNA• It is the 1/10 of 1% that
makes us different from each other
Scientists know where these places are, and look there to determine a DNA profile
DNA is unique
~98% of the DNA in a human cell ~98% of the DNA in a human cell does not code for any proteindoes not code for any proteinSome of this noncoding DNA is tandem repeats:tandem repeats:•The same DNA pattern repeated over and over
•For example: ACACACACAC –or- GTCGTCGTCGTC
Noncoding DNA
How many repeats there are in the DNA How many repeats there are in the DNA varies from person to personvaries from person to personFor example, suppose chromosome 17 had a tandem repeat of ATCGATCGATCG• Some people would have 3 repeats of that sequence
• Some people would have 4 repeats of that sequence
• Some people would have 11 repeats of that sequence, and so on
Noncoding DNA
Scientists use the tandem repeats in a Scientists use the tandem repeats in a person to create a DNA profile or person to create a DNA profile or “fingerprint” “fingerprint”
Noncoding DNA
Step 1:•Get a DNA sample (make copies of it in a lab)
Create a DNA profile (DNA fingerprint)
Step 2: cut the DNAcut the DNA using restriction using restriction enzymesenzymes
Create a DNA profile (DNA fingerprint)
Step 3: sort the DNA fragments sort the DNA fragments by size using by size using gel electrophoresisgel electrophoresis
1. DNA is cut with restriction enzymes2. The cut DNA is placed on a gel3. Electric current runs through the gel4. DNA is negative and moves toward the
positive charge (opposites attract)5. The pieces of DNA separate by size;
smallest move farthest
Create a DNA profile (DNA fingerprint)
Step 4: compare the DNA to the “Suspect” DNA• Scientists typically look at 13 Scientists typically look at 13 different tandem repeat areas different tandem repeat areas (loci)(loci)
•Looking at only 1 does not tell us very much; many people could have that one repeat
•Looking at 13, and being a match for all 13, tells us a lot
• Being a perfect match for all 13 Being a perfect match for all 13 has a chance of 1 in 100 billion has a chance of 1 in 100 billion (there are 7 billion people on the (there are 7 billion people on the planet)planet)
Create a DNA profile (DNA fingerprint)
DNA fingerprinting labVictim – AsuzenaS1: YulissaS2: EricaS3: RajvirS4: HarinderS5: Hernan
Determinerestriction fragmentsizes
• Create standard curve using DNA marker
• Measure distance traveled by restriction fragments
• Determine size of DNA fragments
Identify the relatedsamples
Analysis of Stained Gel
Electrical current carries carries negatively-charged DNA negatively-charged DNA through gel towards through gel towards positive (red) electrodepositive (red) electrode
Agarose Electrophoresis Loading
Power Supply
Buffer
Dyes
Agarose gel
•Agarose gel sieves DNA fragments DNA fragments according to sizeaccording to size– Small fragments Small fragments
move farther than move farther than large fragmentslarge fragments
Agarose Electrophoresis RunningElectrophoresis Running
Power Supply
Gel running
What are we doing overall in this lab?
So…