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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)

http://www.phschool.com/atschool/phbio/gel_electrophoresis/index.html

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…