Lab practical 2 Flashcards
Outline of Lab 2?
- Isolate genomic DNA from swab’s cheek cells for mother, child, and both alleged fathers.
- PCR amplify all 15 STR’s (multiplex PCR) using fluorescent primers.
- Separate and detect PCR products using capillary electrophoresis.
- Genotype all individuals
- Perform genotype exclusion analysis
- Calculate combined paternity index (CPI)
- Convert CPI to probability of paternity
- Call paternity using steps 5-7
What makes a good DNA marker? Why not blood types?
Short tandem repeats (STR’s) make good DNA markers because it is neutral, there is equal frequency in a population, all humans have it, and it is easier to PCR amplify. Blood type does not make a good DNA marker because about 1/1000 people have the same blood type.
PCR reaction components?
- Template DNA: target for DNA amplification
- Primers (bind to compliment of template DNA flanking the target region for amplification)
- DNA polymerase for DNA synthesis
- dNTP’s (incorporated into new DNA)
- Buffers/salts to optimize enzyme activity
PCR Cycle?
- Denature/melt double stranded DNA into single stranded DNA at 94 degrees Celsius (near boiling)
- Anneal (hybridize) primers to complimentary template sequence
- Extend template (DNA synthesis)
- Repeat steps 1-4 (about 30 times)
How are primers designed? Why must we know melting point?
Primers must be located on opposite ends of the STR locus so that they can extend across the STR once PCR amplification starrs. PCR amplicons will be displayed as fluroescent tags every time a PCR product is detected. It is important to know the melting point of primers because it is necessary for multiplex PCR (all 16 primers must have similar annealing temperature)
Definite and explain multiplex PCR. How can so many primers be in the same tube?
Multiplex PCR is the use of more than one primer set within a single PCR reaction to amplify different loci simulataneously. As long as all 16 primers have similar annealing temperatures, they will be in the same reaction tube. 15 target STR’s the other targets amelogenin.
Describe separation and detection of PCR products by capillary electrophoresis. Why is it better than agarose gel electrophoresis?
If the PCR products are non-overlapping from loci, then the same flurorescent label can be used. The fluorescent label should be changed if the product is expected to cross at the same time.
Capillary electrophoresis is better than agarose gel electrophoresis because the sample can run at higher voltages and will be much quicker. Capllary gels can be loaded automatically as well, making it much more time and money efficient.
What do the axes on the capillary electropherogram refer to? How do you read it?
X-axis: time since injection
Y-axis: fluorescence amount
When y-value increases, PCR product is seen
How is the amelogenin gene used for sex typing?
This gene is used for sex typing because it is in the X and Y chromosomes, which determine sex.
How do you calculate paternity index and combined paternity index? How do you calculate probability of paternity? When are you confident the alleged father is the biological father?
Paternity index: probability of alleged father giving child OPA/probability of random man giving child OPA
Combined paternity index (CPI): product of each individual paternity index tested at each loci
Probability of paternity: CPI/CPI+1
Paternity can be assigned when a fathers probability of paternity is a over 99.9%
What are the difference when calculating an identity match versus paternity?
An identity match is based on diploid analysis and is calculated using the Hardy Weinberg equation (p^2 + 2pm = 1). A paternity test only requires the OPA while an identity match is every single allele at each loci.
Why do you need a standard for capillary electrophoresis? How is it used to detect STR?
A standard for capillary electrophoresis allows quantification or PCR products, becoming visible when the fluorescence rises above that standard value.
How do we perform genotype exclusion analysis? If one aSTR does not match up, does that eliminate him from being biological father?
Genotype exclusion analysis is performed by determining the alleles of the mother and child and figuring out which allele is the obligate parental allele (OPA). If an alleged father doesn’t have the OPA, he can be excluded from being the biological father.
