Molecular Diagnostics Flashcards
Southern Blot
DNA
Northern Blot
RNA
Western Blot
Protein
RFLPs
Restriction Fragment Length Polymorphisms
Restriction enzymes
Restriction ENDOnucleases, cut from inside DNA
Leave sticky ends, bacterial protection from bacteriophage
Common Restriction Enzymes
EcoR1, BamH1, HindIII
Gel Electrophoresis
Organizes DNA strands by size of fragments
Sticky ends can be joined together
Can combine DNA from different organisms into plasmids
PCR
Greatly accelerates construction of human genomic DNA library, polymerase chain reaction
PCR Mechanism
- Apply region of DNA to be amplified
- Separate the DNA strands and anneal primers
- DNA synthesis
- Repeat
**Need to add excess nucleotides, DNA polymerase, Taq
Thermus aquaticus
Taq: heat stable DNA polymerase
Thermal cycler
Able to denature, anneal and elongate automatically, PCR machine
Genomic PCR and RT-PCR
- Genomic PCR: amplify DNA
- RT-PCR: reverse transcriptase RNA to cDNA
qRT-PCR: quantitative, how much RNA is there?
DNA travels and grows from 5’ to 3’ direction
C-G (3hb) and A-T (2hb)
Halt DNA synthesis (for sequencing)
Take out OH, make dideoxyribonucleoside (ddATP, ddTTP, ddCTP, ddGTP)
Prevents strand extension at 3’
Fluorochrome
Nucleotide marker, allows for visualization of bands
Southern Blot in detail
- Run DNA on gel
- Denature – transfer to nitrocellulose or nylon membrane
- Can hybridize with radioactive probe to visualize
- Visualize on X-ray
Northern Blot and Western Blot
- Northern Blot: RNA
2. Western Blot: Protein, probe with 1’ and 2’ antibody
Microarrays
- Used to make a DNA profile of a patient, use gene chips
- Normal DNA tagged red
- Tumor DNA tagged green
- Equally normal/tumor = yellow
Can use for diagnostics and to determine treatment
Progression of tumor is correlated with change in gene expression
Can be visualized using microarrays
If tumor genes are being highly expressed
Then can go with aggressive treatment, if not then no
