Week 9 Flashcards
Molecular techniques are highly sensitive methods used to ______ genetic abnormalities and ______ disease progression
detect, monitor
In RNA what base replaces T (Thymine)?
U (Uracil)
What are the 4 base pairs in DNA?
Guanine & Cytosine
Adenine & Thymine
3 base sequences that code for amino acids are called what?
Codons
- A pairs with ____
* G pairs with _____
- A pairs with T
* G pairs with C
How many different amino acids are there and how many different tRNAs
- 20 different amino acids
* 20 different tRNAs
This is a tumour suppressor protein that signals damaged DNA
p53
3 prime is what group and 5 prime is which group?
3' = OH Group 5' = Phosphate group
DNA Synthesis always proceeds:
• Reading __’ to __’ direction
• Synthesis __’ to __’ direction
- Reading 3’ to 5’ direction
* Synthesis 5’ to 3’ direction
Millions of copies of a target area will be made. Specific primers and probes are needed. This is describing what?
PCR - polymerase chain reaction
In addition to positive and negative controls why is a “No DNA” control used?
To exclude contamination
After 30 cycles of PCR amplification how many copies of the target sequence are created?
One billion
Note: A billion copies is enough to
visualise via electrophoresis
What is more stable and easier to work with DNA or RNA?
DNA more stable and easier to work with than RNA
Heat insensitive DNA polymerase from Thermus aquaticus is referred to as what ?
Taq polymerase
PCR master mix contains what 2 things?
Taq polymerase & Nucleotides
What are the 3 steps of the PCR amplification protocol?
Denaturation
Annealing (primers)
Elongation
Briefly describe denaturaiton (include temp)
~95C strands separate but not destroyed
Briefly describe annealing (include temp)
40 – 60C
primers(short DNA molecules) bind to flanking regions of the target DNA
Briefly describe elongation (include temp)
72C TAQ polymerase (a heat resistant bacteria) adds nucleotides to bound primers
How many nucleotides per second added to 5’ end to create amplicon
1000
Primer specificity is critical so that ______ represents area of interest
amplicon
Why is a positive control in PCR used?
To ensure target is being amplified
What does the negative control in a PCR lack?
• Lacks target sequence
DNA has a net negative charge due to what?
nucleic acid phosphate groups
What is the ladder control in gel electrophoresis?
Control containing different size DNA
fragments of known mass
Run alongside other controls and test
samples
Is Standard PCR Qualitative or Quantitative
Qualitative
What does Real-time PCR measure
Measures rate of amplification of target
Quantitative
• Viral load
• Tumour load or minimal residual disease
What is this process called: Recognise and bind to specific nucleotide sequences, Cut the sequence at that point
Restriction endonucleases or restriction enzymes
What does restriction endonucleases produce?
Restriction fragments
• 4 to 15 nucleotides long
What is this process called: Technique used to determine the order in which nucleotides are
arranged in a nucleic acid sequence
DNA sequencing
