Learning Outcomes - Week 2 - Screening for Single Gene Defects

Question 1

Principles of conventional PCR
1. the three phases during PCR reactions and

2. The PCR components
3. How many copies theoretically possible in conventional PCR?

Answer 1

1. Phases are: 1. denaturation 2. annealing 3. extension (see image for details on each)
2. DNA sample, primers, nucleotides, Taq polymerase, mix buffer, PCR tube
3. 2^35 cycles producing 34 billion copies

Question 2

Principles of multiplex PCR
e.g., amplification of different alleles at the same time

Answer 2

Multiplex PCR is the simultaneous detection of multiple targets in a single reaction well, with a different pair of primers for each target. This technique requires two or more probes that can be distinguished from each other and detected simultaneously

(additional info from slide in image)

Question 3

Principles of reverse transcription PCR
e.g., RNA is used as a template to generate _______

1. What is the main difference between RT-PCR and PCR?
2. T/F: RNA use for gene analysis and diagnostic assays has advantages over DNA. List three reasons why
3. How is RNA different to DNA?
4. Converting mRNA to stable cDNA - explain the process

Answer 3

cDNA

1. The main difference between real-time and reverse transcriptase PCR is that real-time PCR gives faster and more detailed results and helps to quantify nucleic acids, whereas RT-PCR helps to detect and amplify cDNA
2. True - a) Doesn’t deal with genome complexities, only coding sequence
   b) Measure of gene expression in cell at the time of sample collection
   c) Monitors progression of therapies
3. • Single stranded rather than double stranded
     - Lots of secondary structure in ssRNA
4. See image

Question 4

Limitations of above types of PCR:
e.g., most importantly: not quantitative, only end-point analyses

Answer 4

Question 5

Principles of real time PCR
e.g., most importantly: it is quantitative; focus on exponential phase

1. What does it measure and how?
2. What are the advantages?
3. What is it also called?
4. What are the three phases?
5. Describe the exponential phase (5 points)
6. Explain the basic principles in terms different Cq values, and what the final PCR product looks like
7. Describe real time PCR quantitation (two approaches used are?), (relative quantitation?)

Answer 5

1. Directly measures and quantifies PCR product as it is produced in real time
   - Amplification plot generated as PCR reaction progresses
   - Fluorescent dyes used for signal generation
   - Cycle at which fluorescence reaches the threshold (Cq) is used to compare the samples not the end-point
2. Advantages
   - Highly sensitive as detects 2-fold changes in target molecule […]
   - Small amount of sample required
   - High throughput capacity
   - Expression of several genes can be measured in one tube
3. Also called qPCR, qRT-PCR:
   - Confusion with reverse transcriptase PCR – also called RT PCR
4. Exponential:
   * Exact doubling of product*
   * Rx is very specific and precise
   * Linear:
   – Rx components become
   limited
   – Efficiency decreases
   * Plateau:
   – Rx has stopped.
   – No more product is being made.
   – End-point detection analysis
5. Exponential phase
   * Exact doubling of product
   * Early detection of product limited by machine sensitivity
   * However: Point of product detection is a measure of initial
   molecule concentration in sample
   * Concentration of target DNA in sample can be extrapolated
   from an arbitary reference point (aka crossing threshold)
   * Approach forms basis of analysis in real time PCR
6. See image
7. Two approaches used:
   * Absolute quantitation
   * Absolute quantity is derived by comparing unknown sample to
   a standard curve (biochemistry)
   * Standard curve created from reference samples with known
   […] of target sequence
   * Known copy number in reference samples is plotted against C q
   * C q of unknown sample compared to standard curve to
   extrapolate gene copy number
   * Relative quantitation
   * Compares target to internal reference/house-keeping gene
   (e.g., β-actin) in same sample

Question 6

Detection of mutations:

1. RFLP (end-point analysis): Restriction enzyme digestion of PCR product (how does it detect?
2. Real time PCR: Can identify number of copies. Explain

Answer 6

1. In the RFLP/PCR (RFLP, restriction fragment length polymorphism) approach of ‘genotypic’ mutation analysis base pair changes, small deletions and insertions are measured which are located in a restriction enzyme recognition sequence and render this site resistant to cleavage by the corresponding endonuclease
2. Quantitative real-time PCR is PCR visualized in real time by the use of fluorescent or intercalating dyes used to measure gene expression or gene quantification including including contiguous gene deletions or duplications. A simple method is described to quantify DNA copy number from human samples

Question 7

Answer 7

f.

(All of the mentioned components are important for a PCR to work, except DNA ligase, which is used in cloning procedures in order to ligate (‘connect’) compatable DNA ends together)

Question 8

(Example of a question that is very detailed and will not be asked)

Name 3 clinical applications of qPCR.

Answer 8

Multiple research applications based on qPCR have been developed. These include gene expression analysis, genotyping, allelic discrimination, single nucleotide polymorphism (SNP) detection, microbe and pathogen detection, species abundance quantification and many others