Housekeeping

1
Housekeeping

• Exam III Friday!
• Review session Wednesday 5 pm (Sc278)
• Same time frame (will be here from 9am to 2
  pm-NEED TO BE DONE BY 2 PM)
• Covers Ch13, Ch12, Ch8, Ch9
• Cancer assignment due May 14th

2
Advantages of RAPD

• SIMPLE and FAST
• No sequence identification necessary!!
• Good for initial genome scan
• unknown organism

3
Detection of DNA polymorphisms

• RFLP
• RFLP PCR
• PCR Southern
• RAPD

4
Detection of polymorphisms REVIEW

• Several different kinds of polymorphisms
• bp substitutions
• mini/microsatellites
• deletions, additions, etc..
• Several different methods to test for
  polymorphisms
• RFLP
• RFLP PCR
• PCR Southern
• RAPD
• Several different uses for polymorphisms
• Forensics
• Genotyping (genetic counseling, genetic
  screening, etc..)
• Evolution
• DNA markers for genome mapping

5
No DNA polymorphism but changes in gene
expression result in disease!! How can we
differentiate gene expression?
6
Saga 4Gene expression in cancer cells
Pancreatic cancer
Control
7
The mouse array

• Each circle/spot represents one sequence of DNA
• Each DNA sequence represents one gene
• All on one glass slide!
• 10,000 spots (10,000 genes)

8
When are these genes expressed? When are these
genes turned on/off in mice with cancer compared
with mice with no cancer?
9
The plan

• 1. Isolate mRNA from mice
• 2. Amplify and label mRNA
• 3. Hybridization onto our microarray (how is this
  different from our Southern hybridization?

10
Video demonstration
http//www.bio.davidson.edu/courses/genomics/chip/
chip.html
11
Pancreatic cancer
Mice grown in same conditions
Control
12
Isolate mRNA from mice

• Isolate total RNA
• Extract mRNA from total RNA
• HOW??

THANK YOU MOLECULAR KITS!
13
The plan

• 1. Isolate mRNA from mice
• 2. Amplify and label mRNA
• 3. Hybridization onto our microarray (how is this
  different from our Southern hybridization?

14
How do we amplify pieces of DNA?
15
Reverse transcription-PCR (RT-PCR)
WHY??
1. Make DNA copies of mRNA 2. Denature DNA 3.
Anneal Primers sit onto DNA 4.
Elongation Amplify DNA piece of interest
HOW??
PCR
16
Poly-A tail
Reverse transcriptase Primer (TTTTTTTTTTT...)
Nucleotides
RT
T
G
C
A
G
Create cDNA
17
What is reverse transcriptase??
What is cDNA??
18
Reverse transcription-PCR (RT-PCR)
1. Make DNA copies of mRNA (cDNA) 2. Denature
DNA 3. Anneal Primers sit onto DNA 4.
Elongation Amplify DNA piece of interest
PCR
19
The plan

• 1. Isolate mRNA from mice
• 2. Amplify and label mRNA
• 3. Hybridization onto our microarray (how is this
  different from our Southern hybridization?