Bioinformatics/PCR Lab
How does having a certain genetic marker affect chances of getting brain cancer?
Bioinformatics/PCR Lab
How does having a certain genetic marker affect chances of getting brain cancer?
Bioinformatics/PCR Lab
Important Concepts:• genetic tests (molecular)• genetic marker• sequence (DNA)• RFLP (restriction fragment length polymorphism)
• genetics and disease• greater risk vs. known association• population studies
Bioinformatics/PCR Lab
Sampling of Genetic Tests Available
-Alzheimer’s Disease*-Lou Gehrig’s Disease
(amyotrophic lateral sclerosis)-breast/ovarian/colon cancer*-cystic fibrosis-fragile X syndrome-Huntington’s Disease-sickle cell anemia-Tay-Sachs Disease
*susceptibility test only
Bioinformatics/PCR Lab
Linking Genetics to Multifactorial Disease (greater risk; involves population studies)
-cancer-other current avenues of
research• peripheral arterial disease• type I diabetes• schizophrenia/bipolar disorder• calcium oxalate stone disease• Chron’s disease• ulcerative colitis
Bioinformatics/PCR Lab
How does having a certain genetic marker affect chances of getting brain cancer?
How does having a specific nucleotidesequence in a specific region of DNA affect chances of getting brain cancer?
Bioinformatics/PCR Lab
How do you determine what the nucleotidesequence is in a piece of DNA?
How do you detect differences in nucleotidesequences in a piece of DNA?
How do you determine if these nucleotidedifferences are related to disease risk?
Bioinformatics/PCR Lab
Bioinformatics:• human genome project (link in course website)• 1990-2003• identify all human genes• sequence human chromosomes• make available to all (databases)
Bioinformatics/PCR Lab
Bioinformatics:• human genome project (link in course website)• comparisons of sequence information• within species• across species • other species genomes sequenced
“They’re Sequencing a What?” Science News, 10/9/04, pp. 234-36.
Bioinformatics/PCR Lab
Bioinformatics:• human genome project (link in course website)• comparisons of sequence information• BLAST search results?• specific DNA segment to work with?• “real” interest in this DNA?• why are we using this segment?
Bioinformatics/PCR Lab
General Lab Procedures:1) isolate DNA from hair (“hair digest”)
2) amplify (make many copies of) DNA
from hair using PCR3) cut PCR product with special enzymes• restriction enzymes• “restriction digest”
Bioinformatics/PCR Lab
General Lab Procedures:4) visualize cut PCR product on a gel5) compare patterns (RFLPs)6) follow people around for decades to see who (which RFLP) gets brain cancer!
Bioinformatics/PCR Lab
Specific Lab Procedures:1) isolate DNA from hair (“hair digest”)
hair with root
400l “master mix #1”(buffer + proteinase K)
1 hour at 65oC;10’ at 100oC
Bioinformatics/PCR Lab
Specific Lab Procedures:2) amplify (make many copies of) DNA
from hair using PCR
“hair digest” tubecontaining isolated DNA
PCR tube50l “master mix #2”
transfer 50l
overnight inthermal cycler
Bioinformatics/PCR Lab
Specific Lab Procedures:3) restriction digest of PCR product
PCR tubecontaining “PCR product”(many copies of desired DNA)
15l “master mix #3”(buffer + PalI)
transfer 5l
1 hour at 37oC
Bioinformatics/PCR Lab
Specific Lab Procedures:4) visualize cut PCR product on a gel
“undigested PCR product”
“digested PCR product”
new 1.5ml microfuge tube
transfer 15l
DNA loading dye
transfer 5l
transfer 5l
Bioinformatics/PCR Lab
Specific Lab Procedures:4) visualize cut PCR product on a gel
“undigested PCR product”
“digested PCR product”
Bioinformatics/PCR Lab
Specific Lab Procedures:6) follow people around for decades to see who (which RFLP) gets brain cancer!
RFLP #1 RFLP #2
if people with RFLP #2have higher incidence ofbrain cancer, then RFLP #2becomes a genetic markerfor brain cancer…
Bioinformatics/PCR Lab
PCR: Polymerase Chain Reaction
• method of making many copies (amplifying) of a specific sequence (region) of DNA in vitro (in a test tube)• developed by Kary Mullis, 1983• Nobel Prize in Chemistry, 1993
Bioinformatics/PCR Lab
PCR: Polymerase Chain Reaction
DNA Replicationin vivo (in a cell)
HelicaseSSBsPrimase (RNA primers)DNA polymerasesLigaseNucleotides (dNTPs, rNTPs)DNA template
DNA Replicationin vitro (PCR)
Not neededNot neededPre-made DNA primersDNA polymerase (Taq)Not neededNucleotides (dNTPs)DNA templateThermal Cycler
Bioinformatics/PCR Lab
PCR: Polymerase Chain Reaction
Thermal Cycler: • cycles through 3 different temperatures• 30 times• each temperature = different step of PCR
1)Denaturation (~94oC; 30 seconds)2)Annealing (~50oC; 30 seconds)3)Primer extension (72oC; 1 minute)
Bioinformatics/PCR Lab
PCR: Polymerase Chain Reaction
1)Denaturation (~94oC; 30 seconds)• dsDNA template separates into two ssDNA• cellular function: helicase, SSBs
Bioinformatics/PCR Lab
PCR: Polymerase Chain Reaction
1)Denaturation (~94oC; 30 seconds)2) Annealing (~50oC; 30 seconds)
• pre-made DNA primers H-bond to template• define region to be amplified• one primer for each strand of template• orientation correct with respect to 3’/5’
• cellular function: primase, primer removal, ligase
Bioinformatics/PCR Lab
PCR: Polymerase Chain Reaction
1)Denaturation (~94oC; 30 seconds)2) Annealing (~50oC; 30 seconds)3) Primer extension (72oC; 1 minute)
• Taq DNA polymerase adds nucleotides to 3’OH• starts from primer• cellular function: same
Bioinformatics/PCR Lab
PCR: Polymerase Chain Reaction
• In the olden days…• 3 waterbaths at each temperature• Fresh DNA polymerase added each cycle
• Automation• Thermal cycler controls temperature• Taq DNA polymerase remains stable
• Taq = Thermus aquaticus
Bioinformatics/PCR Lab
PCR: Polymerase Chain Reaction
Animation: www.dnalc.org/shockwave/pcranwhole.html
Bioinformatics/PCR Lab
Restriction Digest
• Restriction enzymes• Restriction digest = fragments of certain length due to specific sequences
Bioinformatics/PCR Lab
Restriction Digest
Recognizes “the” and cuts between h - e
themonkeyandthedonkeyaretherenow (1)
th emonkeyandth edonkeyareth erenow (4)
themonkeyandthedonkeyarethreenow (1)
th emonkeyandth edonkeyarethreenow (3)
Bioinformatics/PCR Lab
Gel Electrophoresis
Separation of molecules
based on their physical/chemical properties
through a matrix
by applying an electric current
Bioinformatics/PCR Lab
Gel Electrophoresis
Separation of moleculesnucleic acids, proteins
based on their physical/chemical properties
through a matrix
by applying an electric current
Bioinformatics/PCR Lab
Gel Electrophoresis
Separation of moleculesnucleic acids, proteins
based on their physical/chemical propertiessize, charge
through a matrix
by applying an electric current
Bioinformatics/PCR Lab
Gel Electrophoresis
Separation of moleculesnucleic acids, proteins
based on their physical/chemical propertiessize, charge
through a matrixagarose, polyacrylamide
by applying an electric current
Bioinformatics/PCR Lab
Gel Electrophoresis
Separation of moleculesnucleic acids (DNA)
based on their physical/chemical propertiessize (length in base pairs - bp)
through a matrixagarose
by applying an electric current
Bioinformatics/PCR Lab
Gel Electrophoresis
Before - samples insample wells
After - samples movedthrough gel
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