Practical Study guide Flashcards
what is the 2+ 4 rule
it describes the melting temperature
for every A/T, add 2 degrees C
for every G/C add 4 degrees C
why do we add only 2 degrees for A/T
Adenine and Thymine only have 2 hydrogen bonds between them so it takes less overall energy to melt off the primers in AT rich regions
why do we add 4 degrees for G/C
Guanine and Cytosine have 3 hydrogen bonds between them which takes more energy to melt off the primers in GC rich regions
What is the PI?
it is the paternity index, it describes the probability that the alleged father gave an allele to a child in comparison to the likelihood that a random man gave the allele to the child
what does the PI help us determine
it helps determine if the alleged father is the biological father and it is not some random man
how do we calculate the PI?
The probability that the AF gave the allele to the child (0.5-1) / the probability that a random man gave the allele to the hild (frequency of allele in general population
if a man is heterozygous, what is the probability that he gave an allele to a child
0.5
if a man is homozygous, what is the probability that he gave an allele to a child
1
what is the combined PI (CPI)
it is the product of PI at all loci tested
how do we calculate the probability of paternity
CPI/ (1+CPI)
what does the probability of paternity help us determine
how probable it is that the alleged man is the father
what are the conditions to assign paternity in the PP
if PP > 99.9% paternity is assigned
what are short tandem repeats?
they are repetitive DNA sequences typically found in introns or non coding sequences of DNA
why are STR’s good markers for identity profiling
they are highly polymetric as different individuals may have different numbers of repeats
the allele frequency of repeats is fairly equal and evenly distributed throughout the population
they are small enough to be evenly be amplified by PCR
how is the amelogenin gene used for sex-typing
the amelogenin gene is is both the X and Y chromosome, but in the X chromosome there is a 6 base pair deletion that makes the subsequent PCR product shorter.
A shorter product will come out of the capillary electrophoresis tube faster and will be detected earlier than the Y chromosome will. This allows a distinct separation between the two peaks.
For individuals that are homozygous for the X chromosome (females), they will have one of these peaks
while individuals that have the X and Y chromosome (males), they will have two peaks that are separated by this 6 base pair deletion
these electropherogram differences allow the gender to be distinguished
how many amelogenin peaks will females have?
one shorter peak for the 6 bp deletion in the X
how many amelogenin peaks will males have
they will have two peaks, the earlier peak will be the shorter X chromosome and the later peak will be the Y chromosome
what is meant by multiplexing? What is one challenge that occurs with the design of multiplexing?
multiplexing is when more than one locus is amplified in a PCR reaction, it requires all primers to have similar annealing temperatures
how can we distinguish between multiplexed PCR products of the same size?
the PCR primers have been fluorescently tagged, so alleles of the same size have been assigned different fluorescent color tags that allow the overlapping alleles to be distinguished from one another
what are the steps to PCR
1) denaturation
2) primer annealing
3) primer elongation
4) cycling
what is the purpose of denaturation
we increase the temperature of the reaction ot break the hydrogen bonds between the double stranded DNA and allow it to become single stranded
what is the purpose of primer annealing
the primers hybridize to their complementary sequence and specify to the polymerase which DNA sequence is supposed to be copied
provides 3’ OH group required by polymerase to synthesize new DNA
qhat is the purpose of primer elongation
elongate primers towards eachother in 5’-3’ direction to create target DNA molecules
what is the purpose of cycling
allows us to repeat the PCR cycle numerous times on each of the newly synthesized target molecules
why is Taq polymerase used in PCR
it is a heat stable polymerase that comes from heat tolerant bacteria that can withstand the higher temperatures involved in PCR
what are the ingredients to PCR
1) DNA template
2) Primers
3) DnTPS
4) DNA polymerase
5) Buffer with magnesium and salts
what is the purpose of having a DNA template
provides a sequence to amplify
what is the purpose of having DNA primers
primers allow targeting the sequence you want to amplify and it provides a 3’OH for the polymerase
what is the purpose of having DnTPS
provides nucleotides for elongating the new DNA target sequences
what is the purpose of having DNA polymerase
synthesizes the new DNA from the primer
what is the purpose of having salts and buffer with magnesiunm
DNA pol requires magnesium
How was the template DNA that we used in the PCR obtained
through cheek swabs from members of the paternity case
what is meant by the melting temperature
it is the temperature where 50% of DNA is double stranded vs single stranded
here primers just barely sit down/ anneal to the template
What are the two properties of primers that influence melting and annealing temperatures
GC/AT rich content: higher GC content leads to higher annealing temperatures due to the presence of three hydrogen bonds
Primer length: longer primers have higher annealing temperatures
how does GC content influence primer melting/annealing temperature
GC rich areas will require higher annealign temperatures due to the fact that guanine and cytosine form 3 hydrogen bonds. These bonds are harder to denature and it requires more energy for primers to reach their melting temperature
how does primer length influence melting/annealing temperatyre
longer primers are more susceptible to non-specific annealing. To avoid this, longer primers typically require higher temperatures to ensure specificity
why do all primers in a given PCR reaction need to have similar melting/annealing temperatures
they must have similar annealing temperatures to ensure that all primers anneal specifically at a given temperature. If you have a primer that has a higher annealing temperature and another that is too low, the higher annealing primer may not anneal specifically
if you wanted to increase the specificity of amplification in a PCR reaction, would you raise or lower the annealing temperature
you would raise the annealing temperature. Primers annealed near their melting temperature will “sit down” specifically on their complementary sequence.
How does capillary gel electrophoresis work
samples are ran through a thin tube that is filled with a polymer that acts as a sieve to separate smaller and larger PCR products by size.
similar to gel electrophoresis, the smaller products will elute from the tube faster and be detected earlier by the electropherogram
why do we use capillary electrophoresis over gel?
it allows us to make smaller separations between PCR products that vary by 1 base pair. This is important because our PCR products may only vary by a few repeats
What are the advantages of capillary electrophoresis (4 reasons)
it allows smaller separations
the diffusion and convection forces are reduced, which allows us to use higher voltages and elute in shorter amounts of time
they can be loaded automatically and are more sensitive which saves both time, money, and the amount of sample required
how do electropherograms distinguish between PCR products
by size and color (fluorescent tags)
what does the Y axis on the electropherogram read
it reads the amplitude of the signal in relative fluorescent units. This indicates the relative amount of DNA in each peak
do PCR products with less or more repeats come out first
Products with less repeats come out first as they move through the column more quickly
how do you tell if a individual is homozygous or heterozygous at a given loci
if they are homozygous, they will only show one peak at a given loci. This means they have the same number of repeats on either chromosome
if they are heterozygous, they will show two peaks at a given loci. This indicates that they have different numbers of repeats on either chromosome
Which amelogenin allele will be detected first during capillary electrophoresis- the one on the X or Y?
the X allele will be detected firsta s it has a 6 base pair deletion
if the mother has the alleles 8, 9 and the child has the alleles 8 and 10, what is the obligate parent allele
10, the mother gave the child the 8 allele
what is being meausred in the paternity index
the probability that the alleged father is the biological father and not some random man
what is being measured by Random Match Probability? what is it used for?
it is used for identity matching. It will tell you the estimated frequency of an STR profile in a population. All the RMP values at each locus are calculated individually and then multiplied
what is the difference between RMP analysis and paternity analysis
RMP analysis is a diploid analysis sued for identity matching in crime investigations. It tells yout he frequnecy of an STR profile in a population
A paternity analysis is a haploid analysis that is used to calculate the probability that an alleged father could have provided all the alleles that a child has from their biological father
