Molecular Diagnostics Flashcards
What are the molecular techniques?
- Restriction enzymes
- Cloning
- Polymerase chain reaction (PCR)
- Restriction fragment length polymorphisms (RFLP)
- Sequencing techniques
- Blotting techniques
- Microarrays
- SNPS
What are restriction enzymes?
Recognize certain sequences (pallindromic) and cut them, restriction endonucleases
What are sticky ends and what is their advantage?
They are…
Can easily be joined together, allows cloning
What are sticky ends and what is their advantage?
They are…
Can easily be joined together, allows cloning
What is PCR and what is its purpose?
Polymerase chain reaction, amplifies the DNA section of interest
What is required to perform PCR analysis?
- Knowledge of sequence of interest for primers
- Excess nucleotides
- DNA polymerase
- Change temperatures: denature, anneal, elongate
What were the initial problems of PCR and how were they solved?
- New DNA polymerase needed for each cycle, solved by Thermus aquaticus bacterium “can tolerate hot temperatures”
- Had to move to a new water bath after each cycle, solved with the thermal cycler
What are the different types of PCR?
Genomic PCR, RT-PCR, Long Range PCR, Multiplex PCR
What is Genomic PCR?
Amplify genomic DNA
What is RT-PCR?
Reverse transcriptase RNA to cDNA then follow with PCR
What is Long Range PCR?
Used for big DNA section
What is multiplex PCR?
Used for running several PCR @ once
How do DNA fragments of different sizes run through agarose gel?
Small DNA fragments run faster
What results can be interpreted with PCR and R.E.
Altered RE recognition site, deleted sequences, inserted sequences
What is dideoxyribonucleoside triphosphate (ddATP) used for?
Stops DNA synthesis to make a fragment of only a certain size
