Download presentation
Presentation is loading. Please wait.
Published byAvice Wells Modified over 9 years ago
1
Bioinformatics/PCR Lab How does having a certain genetic marker affect chances of getting brain cancer?
2
Bioinformatics/PCR Lab How does having a certain genetic marker affect chances of getting brain cancer?
3
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
4
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
5
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
6
Bioinformatics/PCR Lab How does having a certain genetic marker affect chances of getting brain cancer? How does having a specific nucleotide sequence in a specific region of DNA affect chances of getting brain cancer?
7
Bioinformatics/PCR Lab How do you determine what the nucleotide sequence is in a piece of DNA? How do you detect differences in nucleotide sequences in a piece of DNA? How do you determine if these nucleotide differences are related to disease risk?
8
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)
9
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.
10
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?
11
Bioinformatics/PCR Lab General Lab Procedures: 1) isolate DNA from hair ( “ hair digest ” ) 2) amplify (make many copies of) DNA from hair using PCR 3) cut PCR product with special enzymes restriction enzymes “ restriction digest ”
12
Bioinformatics/PCR Lab General Lab Procedures: 4) visualize cut PCR product on a gel 5) compare patterns (RFLPs) 6) follow people around for decades to see who (which RFLP) gets brain cancer!
13
Bioinformatics/PCR Lab Specific Lab Procedures: 1) isolate DNA from hair ( “ hair digest ” ) hair with root 400 l “ master mix #1 ” (buffer + proteinase K) 1 hour at 65 o C; 10 ’ at 100 o C
14
Bioinformatics/PCR Lab Specific Lab Procedures: 2) amplify (make many copies of) DNA from hair using PCR “ hair digest ” tube containing isolated DNA PCR tube 50 l “ master mix #2 ” transfer 50 l overnight in thermal cycler
15
Bioinformatics/PCR Lab Specific Lab Procedures: 3) restriction digest of PCR product PCR tube containing “ PCR product ” (many copies of desired DNA) 15 l “ master mix #3 ” (buffer + PalI) transfer 5 l 1 hour at 37 o C
16
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 15 l DNA loading dye transfer 5 l
17
Bioinformatics/PCR Lab Specific Lab Procedures: 4) visualize cut PCR product on a gel “ undigested PCR product ” “ digested PCR product ”
18
Bioinformatics/PCR Lab Specific Lab Procedures: 5) compare patterns (RFLPs) RFLP #1RFLP #2
19
Bioinformatics/PCR Lab Specific Lab Procedures: 6) follow people around for decades to see who (which RFLP) gets brain cancer! RFLP #1RFLP #2 if people with RFLP #2 have higher incidence of brain cancer, then RFLP #2 becomes a genetic marker for brain cancer …
20
Bioinformatics/PCR Lab Techniques to Know: 1) PCR 2) Restriction Digest 3) Gel Electrophoresis
21
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
22
Bioinformatics/PCR Lab PCR: Polymerase Chain Reaction DNA Replication in vivo (in a cell) Helicase SSBs Primase (RNA primers) DNA polymerases Ligase Nucleotides ( dNTPs, rNTPs ) DNA template DNA Replication in vitro (PCR) Not needed Pre-made DNA primers DNA polymerase (Taq) Not needed Nucleotides ( dNTPs ) DNA template Thermal Cycler
23
Bioinformatics/PCR Lab PCR: Polymerase Chain Reaction Thermal Cycler: cycles through 3 different temperatures 30 times each temperature = different step of PCR 1)Denaturation (~94 o C; 30 seconds) 2)Annealing (~50 o C; 30 seconds) 3)Primer extension (72 o C; 1 minute)
24
Bioinformatics/PCR Lab PCR: Polymerase Chain Reaction 1)Denaturation (~94 o C; 30 seconds) dsDNA template separates into two ssDNA cellular function: helicase, SSBs
25
Bioinformatics/PCR Lab PCR: Polymerase Chain Reaction 1)Denaturation (~94 o C; 30 seconds) 2) Annealing (~50 o C; 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
26
Bioinformatics/PCR Lab PCR: Polymerase Chain Reaction 1)Denaturation (~94 o C; 30 seconds) 2) Annealing (~50 o C; 30 seconds) 3) Primer extension (72 o C; 1 minute) Taq DNA polymerase adds nucleotides to 3 ’ OH starts from primer cellular function: same
27
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
28
Bioinformatics/PCR Lab PCR: Polymerase Chain Reaction Animation: www.dnalc.org/shockwave/pcranwhole.html
29
Bioinformatics/PCR Lab Restriction Digest Restriction enzymes Restriction digest = fragments of certain length due to specific sequences
30
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)
31
Bioinformatics/PCR Lab Gel Electrophoresis Separation of molecules based on their physical/chemical properties through a matrix by applying an electric current
32
Bioinformatics/PCR Lab Gel Electrophoresis Separation of molecules nucleic acids, proteins based on their physical/chemical properties through a matrix by applying an electric current
33
Bioinformatics/PCR Lab Gel Electrophoresis Separation of molecules nucleic acids, proteins based on their physical/chemical properties size, charge through a matrix by applying an electric current
34
Bioinformatics/PCR Lab Gel Electrophoresis Separation of molecules nucleic acids, proteins based on their physical/chemical properties size, charge through a matrix agarose, polyacrylamide by applying an electric current
35
Bioinformatics/PCR Lab Gel Electrophoresis Separation of molecules nucleic acids (DNA) based on their physical/chemical properties size (length in base pairs - bp) through a matrix agarose by applying an electric current
36
Bioinformatics/PCR Lab Gel Electrophoresis Before - samples in sample wells After - samples moved through gel ++
37
Bioinformatics/PCR Lab Gel Electrophoresis -why does DNA migrate to + end? +
38
Bioinformatics/PCR Lab Gel Electrophoresis -size vs. location of DNA bands on gel? +
39
Bioinformatics/PCR Lab Gel Electrophoresis -how do you know size of DNA fragments? + Standards of known DNA size (bp) are loaded onto the gel “ DNA ladder ” 1000 800 600 400 200
Similar presentations
© 2025 SlidePlayer.com. Inc.
All rights reserved.