Basic DNA Analysis Methods Flashcards
This technique permits the production of an enormous number of copies of specific DNA sequences starting with a minute quantity.
Polymerase chain reaction (PCR)
How much template DNA is needed for PCR?
50 ng or less
T/F: After amplification, the corresponding product constitute the bulk of DNA present in the PCR reaction tube.
True
After 25-35 cycles, the target has been, in theory, amplified by?
2^25 - 2^35 fold
This component of PCR is designed to be complementary to the flanking sequences of the target segment of DNA that is to be amplified.
Oligonucleotide primer
Target DNA sequence during PCR is commonly up to ____, and occasionally up to ____.
1 kb; 10 kb
Give an example of a thermostable polymerase.
Taq polymerase
What is the common denaturing temperature for PCR?
95 degrees C
_____ direct repeated cycles or rounds of _____ to produce an _____ in the number of copies of the target sequence.
Primers; localized DNA replication; exponential increase
What are the three basic steps of PCR?
Heat denaturation; primer annealing; strand elongation
This step of PCR renders the DNA sample single-stranded.
Heat denaturation
What are the two common staining dyes for PCR products?
Ethidium bromide; SYBR safe
DNA amplification products can be visualized under ____ following ____ and staining with a DNA stain such as ____ or a less mutagenic compound such as ____.
UV light; gel electrophoresis; ethidium bromide; SYBR safe
To detect small changes such as ____ and small ____ or ____, DNA product will be sequenced by an automatic DNA sequencing strategy.
nucleotide substitutions; insertions; deletions
To detect specific sequence changes, the DNA can be subjected to an appropriate ____ and the resulting fragments separated and visualized by ____ and ____, respectively.
restriction enzyme digest; gel electrophoresis; DNA staining
What is the restriction site sequence for EcoR1?
5’-G/AATTC-3’
What is the restriction site sequence for Taql?
5’-T/CGA-3
What is the restriction site sequence for Smal?
5’-CCC/GGG-3’
Adaptations of PCR include:
quantitative fluorescent PCR (QF-PCR); allele-specific PCR such as in amplification - refractory mutation system; triplet repeat - primed PCR; multiplex ligation -dependent probe amplification
____ are reactions that use DNA cleavage enzymes, which will cut at specific DNA sequences.
Restriction digests
The enzyme’s target sequence can be detected by testing the ability of a ____ to cut a patient’s at that target sequence.
restriction enzyme
The increasing availability and efficiency of ____ and ____, the use of restriction digests has become much less common than previously.
DNA sequencing technology; allele-specific PCR
