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:5) compare patterns (RFLPs)

RFLP #1 RFLP #2

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

Techniques to Know:1) PCR2) Restriction Digest3) Gel Electrophoresis

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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Bioinformatics/PCR Lab

Gel Electrophoresis-why does DNA migrate to + end?

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Bioinformatics/PCR Lab

Gel Electrophoresis-size vs. location of DNA bands on gel?

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Bioinformatics/PCR Lab

Gel Electrophoresis-how do you know size of DNA fragments?

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Standards of knownDNA size (bp) are loadedonto the gel “DNA ladder”1000

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