AAB MOLECULAR DIAGNOSTICS STUDY
SHEET 2026 DETAILED QUESTION SERIES
◉ Characteristics of an ideal primer.
Answer: -melting temperature (Tm) between 55 and 65C (usually
corresponds to 45-55% G +C for a 20-mer)
-absence of dimerization capability
-absence of significant hairpin formation (usually >3 bp)
-lack of secondary priming sites in the template
-low specific binding at the 3' end, to avoid mispriming
◉ Qualities of Endpoint PCR (Classical PCR).
Answer: -analysis of amplicon products occurs after PCR reaction
has run to completion (static)
-data collected at the plateau phrase of PCR reaction
-usually performed using standard thermocycler followed by
electrophoresis and detection techniques
-EtBr visualization common
-may use single of primers or multiplexed (2 or more primer sets)
◉ RT-PCR data analysis.
Answer: -when target copy # vs. # of cycles is graphed
,-an exponential curve where number of target copies= 2N
◉ What occurs in the exponential phase of classical PCR.
Answer: accumulation of fluorescence is inversely proportional to
the amount of starting template
◉ Threshold cycle (CT) (Classical PCR).
Answer: PCR cycle at which sample fluorescence crosses an
established threshold amount of fluorescence
◉ Melt allele (Classical PCR).
Answer: specific RT-PCR products over defined temperature range
◉ Reverse transcriptase PCR (RT-PCR).
Answer: -utilizes mRNA as initial input
-synthesizes complementary DNA (cDNA) which is template for DNA
reaction
-primers for reverse transcription
-can be 1 or 2 reaction setup
◉ Types of Primers for RT-PCR.
Answer: -target specific
, -random hexamers
-oglio dT
◉ Target specific primers (RT-PCR).
Answer: only amplifies RNA with desired sequence
◉ Random hexamers primers (RT-PCR).
Answer: 6-10 base long random sequence primers that amplify all
RNAs in sample
◉ Oligo dT primers (RT-PCR).
Answer: 18 base long single stranded polyT sequences that will
amplify only mRNA with polyA tails
◉ Two sets of primers are utilized to ampify a single target
(Nested/Semi-Nested PCR).
Answer: first round
second round
◉ First round primers (Nested/Semi-Nested PCR).
Answer: target long product within genomic cDNA
SHEET 2026 DETAILED QUESTION SERIES
◉ Characteristics of an ideal primer.
Answer: -melting temperature (Tm) between 55 and 65C (usually
corresponds to 45-55% G +C for a 20-mer)
-absence of dimerization capability
-absence of significant hairpin formation (usually >3 bp)
-lack of secondary priming sites in the template
-low specific binding at the 3' end, to avoid mispriming
◉ Qualities of Endpoint PCR (Classical PCR).
Answer: -analysis of amplicon products occurs after PCR reaction
has run to completion (static)
-data collected at the plateau phrase of PCR reaction
-usually performed using standard thermocycler followed by
electrophoresis and detection techniques
-EtBr visualization common
-may use single of primers or multiplexed (2 or more primer sets)
◉ RT-PCR data analysis.
Answer: -when target copy # vs. # of cycles is graphed
,-an exponential curve where number of target copies= 2N
◉ What occurs in the exponential phase of classical PCR.
Answer: accumulation of fluorescence is inversely proportional to
the amount of starting template
◉ Threshold cycle (CT) (Classical PCR).
Answer: PCR cycle at which sample fluorescence crosses an
established threshold amount of fluorescence
◉ Melt allele (Classical PCR).
Answer: specific RT-PCR products over defined temperature range
◉ Reverse transcriptase PCR (RT-PCR).
Answer: -utilizes mRNA as initial input
-synthesizes complementary DNA (cDNA) which is template for DNA
reaction
-primers for reverse transcription
-can be 1 or 2 reaction setup
◉ Types of Primers for RT-PCR.
Answer: -target specific
, -random hexamers
-oglio dT
◉ Target specific primers (RT-PCR).
Answer: only amplifies RNA with desired sequence
◉ Random hexamers primers (RT-PCR).
Answer: 6-10 base long random sequence primers that amplify all
RNAs in sample
◉ Oligo dT primers (RT-PCR).
Answer: 18 base long single stranded polyT sequences that will
amplify only mRNA with polyA tails
◉ Two sets of primers are utilized to ampify a single target
(Nested/Semi-Nested PCR).
Answer: first round
second round
◉ First round primers (Nested/Semi-Nested PCR).
Answer: target long product within genomic cDNA
