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Catalyst: 3-17Catalyst: 3-17
What is gene therapy? How can it help What is gene therapy? How can it help people?people?
Genetic DiseasesGenetic Diseases
Medical conditions caused by an Medical conditions caused by an error in a person’s genetic materialerror in a person’s genetic material
Some Show as Birth DefectsSome Show as Birth Defects Others do not become evident until Others do not become evident until
childhood or adult life. childhood or adult life. Can be mild (color blindness) to life Can be mild (color blindness) to life
threateningthreatening
Gene Therapy• Gene therapy refers to treating genetic disorders
by correcting a defect in a gene or by providing a normal form of a gene.
This Little Light This Little Light of Mine:of Mine:
Transform bacteria with a Jellyfish
gene to make them glow
Aequorea victoria: Source of “glowing gene” for this experiment
Jellyfish Gene put into Other CrittersJellyfish Gene put into Other Critters
HOW DOES THIS HAPPEN???
DNA TECHNOLOGY!!!
• Recognize specific base sequences in DNA• Cut DNA at those recognition sites
Restriction Enzymes
Recombinant DNA: combination of DNA from 2 or more sources
Put Recombinant into bacteria cells and clone them… then put them
where you want them!!!
SCIENCE IN THE NEWS: Seg 7
• What is manic depression?
• What is its connection to the human genome project?
• How might the discovery of a genetic cause for manic depression help patients like Kay Redfield Jamison, the woman featured in the segment?
• According to Francis Collins, the second scientist interviewed, why are scientists excited about the Human Genome Project?
STUDY GUIDES• In Groups• Work on page 64 Diagram first off• Then work on the rest of that section
DNA Fingerprinting
Determination of an individual’s unique collection of DNA restriction fragments
Collect Tissue Sample
How to do DNA FingerprintingThe Big Picture
>1000 cells
RFLP / Southern blot PCR AnalysisRFLP / Southern blot
>20 cells
RFLP Analysis
RFLP – Restriction Fragment Length Polymorphism; for related DNA molecules, a difference in DNA fragment sizes after restriction enzyme digestion– Difference results from presence of different
DNA sequences– Certain regions of genome are highly variable
AGATCTWild-type allele
Mutant allele
TCTAGA
A single nucleotide change can make a difference
AGAGCT
TCTCGA
Restriction site
Not a restriction site
Example: Sickle-cell allele destroys an MstII site
Need to Analyze only a Small Fraction of Genome
• Human genome is too big to analyze:
• 3 x 109 base pairs 65,536 bp between cuts
= ~46,000 bands
• Most regions of genome are not suitable:
• 99.9% of DNA sequence is same from one person to the next
• Solutions:
• Limit analysis to a few genomic regions
• Focus on regions which are highly variable
How to Focus on Specific Regions of Genome
Need a probe:A short single stranded DNA which is complementary to the region of interest
CAGTATACACAAGTACCGTACCTGGCTCAGTTATACGCCGA
A probe will base pair to the region of interest
GTCATATGTGTTCATGGCATGGACCGAGTCAATATGCGGCT:::::::::::::::::::::::::::::::::::::::::
ATGGCATGGACC::::::::::::
probe
Southern Blotting
Simple Tandem RepeatsSimple Tandem Repeats(STRs)(STRs)
Simple Tandem Repeats(STR’s)
STR – region of DNA containing tandem copies of di-, tri- or tetranucleotide repeat units.
Examples:Dinucleotide repeats: GTGTGTGTGTGT……
Trinucleotide repeats: ACGACGACGACG……
Tetranucleotide repeats:TATCTATCTATC……
More on STRs
• Number of repeats varies greatly between individuals
• STRs make up 10-15% of the mammalian genome
• STRs are also called “microsatellites”
• STRs are “junk DNA”
Regions of Chromosome Analyzed for DNA
Fingerprinting Often Contain STRs
ACTACTPerson 1 ACTACT
100 ACTrepeats
EcoRI EcoRI
Person 2 ACTACTACTACT
400 ACTrepeats
EcoRI EcoRI
EcoRI fragment from Person 2 is 900bp longer than in Person 1
DNA SynthesisDNA Synthesis
Separatestrands
DNA Replication is Semi-Conservative
AGTCAG
TCAGTC
------
5
53
3
TCAGTC
AGTCAG
5
53
3
AGTCAG
AGTC
------
------
AGTC
TCAGTC
5
53
3
5
5
3
3
AGTCAG
TCAGTC
------
------
AGTCAG
TCAGTC
5
53
3
5
53
3
Add correct bases
Building a Strand of DNA
• DNA polymerase – enzyme that synthesizes DNA
• DNA polymerase can only add nucleotides to the 3´ end of a strand
• DNA polymerase cannot build a new strand without a primer
DNA Polymerase Needs a Primer
3´ 5´ss DNA
+Nucleotides (dNTPs)
+DNA polymerase
=No DNA synthesis
No DNA synthesis
DNA Polymerase Needs a Primer
3´ 5´ss DNA
+Nucleotides (dNTPs)
+DNA polymerase
= DNA synthesis
5´ 3´primer
DNA Polymerase Needs a Primer
3´ 5´ss DNA
+Nucleotides (dNTPs)
+DNA polymerase
= DNA synthesis
5´primer
Polymerase Chain Reaction Polymerase Chain Reaction (PCR)(PCR)
Collect Tissue Sample
How to do DNA FingerprintingThe Big Picture
>1000 cells
RFLP / Southern blot PCR AnalysisPCR Analysis
>20 cells
PCR
Purpose – Quickly make many copies of a region of a DNA molecule
Method – Multiple rounds of DNA replication
Components in PCR reaction – Target DNA, nucleotides, DNA polymerase, and primers
Temperature cycling – DNA replication controlled by temperature…
Temperature Cycling in PCR
Temperature cycling – PCR process uses a machine (thermocycler) in which PCR reaction goes through ~30 cycles of three different temperature changes:
~95ºC – Melting temperature
50-65ºC – Annealing temperature
72ºC – Extension temperature
Polymerase chain reaction (PCR) analysis1). primers are designed to flank the region to be amplified in target DNA2). primers are annealed to denatured DNA3). DNA is synthesized using Taq polymerase (from Thermus aquaticus)4). primers are annealed again and the process is repeated through
20-30 cycles, geometrically amplifying the target sequence5). DNA is analyzed by gel electrophoresis
1).
2).
3).
4).
DNA Technology CSI style
DNA FingerprintingDNA Fingerprinting
Determination of an individual’s unique collection of DNA restriction fragments
Collect Tissue Sample
How to do DNA FingerprintingThe Big Picture
>1000 cells
RFLP / Southern blot PCR AnalysisRFLP / Southern blot
>20 cells
PCR
Purpose – Quickly make many copies of a region of a DNA molecule
Method – Multiple rounds of DNA replication
RFLP AnalysisRFLP Analysis
RFLP – RFLP – RRestriction estriction FFragment ragment LLength ength PPolymorphismolymorphism
-Cut DNA fragments into different sizes w/ -Cut DNA fragments into different sizes w/ restriction enzymerestriction enzyme
-look for matches-look for matches
DNA specimen is extracted from blood or other DNA specimen is extracted from blood or other tissuetissue
Restriction enzymes cut up DNARestriction enzymes cut up DNA DNA fragments are placed in wells made on gelDNA fragments are placed in wells made on gel Electric current is run through gelElectric current is run through gel Fragments separate by sizeFragments separate by size Single chains of separated DNA fragments are Single chains of separated DNA fragments are
blotted onto filter paperblotted onto filter paper Probes bind to complementary DNA fragments Probes bind to complementary DNA fragments
in the samplein the sample Visible bands form on exposed photographic filmVisible bands form on exposed photographic film Film is developed to reveal a DNA fingerprintFilm is developed to reveal a DNA fingerprint
Fingerprint AnalysisFingerprint Analysis
Let’s make some fingerprints!!!Let’s make some fingerprints!!!
Fingerprint TerminologyFingerprint Terminology ridge ending - a ridge that ends abruptly; ridge ending - a ridge that ends abruptly; bifurcation - a single ridge that divides into two ridges; bifurcation - a single ridge that divides into two ridges; lake or enclosure - a single ridge that bifurcates and lake or enclosure - a single ridge that bifurcates and
reunites shortly afterwards to continue as a single ridge; reunites shortly afterwards to continue as a single ridge; short ridge, island or independent ridge - a ridge that short ridge, island or independent ridge - a ridge that
commences, travels a short distance and then ends; commences, travels a short distance and then ends; dot - an independent ridge with approximately equal dot - an independent ridge with approximately equal
length and widthlength and width spur - a bifurcation with a short ridge branching off a spur - a bifurcation with a short ridge branching off a
longer ridge longer ridge crossover or bridge - a short ridge that runs between two crossover or bridge - a short ridge that runs between two
parallel ridges. parallel ridges.
Draw and label Draw and label these three on these three on
your thumbprintyour thumbprint
ridge ending - a ridge that ends abruptly; ridge ending - a ridge that ends abruptly; bifurcation - a single ridge that divides into bifurcation - a single ridge that divides into
two ridges; two ridges; lake or enclosure - a single ridge that lake or enclosure - a single ridge that
bifurcates and reunites shortly afterwards bifurcates and reunites shortly afterwards to continue as a single ridge; to continue as a single ridge;