Exp 2 Quizzes Flashcards
STR locations that are amplified for the CODIS system (combined DNA index system) are found on different chromosomes for each unique STR.
true
Which of the following is true regarding Multiplex PCR?
more than one primer set is used in the same reaction
more than DNA locus (location) is targeted for amplificaiton.
A single set of primers within the reaction can amplify multiple regions of the genome
there can be two different PCR products for each STR location in the genome if the parent is heterozygous
More than one primer, More than one Gene locus, two different PCR products for each STR location
Which of the following is true regarding capillary electrophoresis?
it is better at separating DNA than agarose gel electrophoresis
it uses the fluorescence on the primers that integrate into PCR products to detect the DNA
it separates DNA based on size where larger DNA travels faster in the capillary than smaller sized fragment
it is better at separating DNA than agarose gel electrophoresis
it uses the fluorescence on the primers that integrate into PCR products to detect the DNA
Identical twins will have the same exact STR profile when their electropherograms are analyzed.
true or false
false
When performing the genotype exclusion analysis, the obligate paternal allele is the STR repeat allele that the mother did not give to the child.
Group of answer choices
true
The paternity index for any one STR locus is calculated based on the Hardy Weinberg equation to account for homozygote and heterozygote frequencies.
false
The Combined Paternity Index is calculated simply by taking the product of all individual PI’s (Paternity Indices) at all loci tested.
true
Identity matching that is used for Crime Scene Investigations (CSI) is based on examining whether the suspect’s alleles at ALL STR loci match the individual in question.
Group of answer choices
true
What are the steps to this experiment
1.) extract genomic DNA
2.) Determine DNA concentration
3.) Set up multiplexed PCR
4.) Separate PCR products with capillary electrophoresis
5.) Analyze PCR
What is the purpose of PCR
to make billions of copies of a specific DNA sequence of interest
Does PCR eliminate the rest of the DNA when it amplifies the specific target sequence?
n, but it makes so many copies of the specific sequence that there is only a minimal amount of interference from the rest of the DNA
What biological process does PCR imitate
it imitates DNA replication but only amplifies short highly specific stretches of DNA
What are the 4 steps to PCR
1) Denaturation 2) primer annealing 3) primer extension 4) cycling
What defines the sequences of DNA that are amplified
the primers used in the reaction
What is the purpose of the denaturing step
When we denature, we are separating the double-stranded DNA and exposing the single-stranded regions to the DNA polymerase
In PCR, how do we denature the DNA
we use heat
What temperature is the denaturing step of PCR
92-94 °C
What is the purpose of annealing the primers
the primers need to find their single-stranded complementary sequences and hybridize
what do the primers do?
the primers specify which region the polymerase needs to amplify and provides a 3’OH
At what temperature do primers have the greatest specificity?
at the melting temperature
What occurs at the melting temperature
the primers just barely “sit down” on its complementary sequence without melting off
What types of primers have lower melting temperatures?
AT rich primers are less stable and tend to melt off more easily than GC rich primers so they have lower melting temperatures
When assigning melting temperatures, how many degrees do we assign for each A/T? what about G/C?
For each A/T we add 2
For each G/C, we add 4
What occurs during the primer extension step
DNA polymerases recognize the 3’ end of the polymerases ad begins extending the primers TOWARDS each other across the target sequence
what directions are the primers extended?
towards eachother
what is the most favorable temperature for the extension step?
72 °C
What occurs during the cycling step
the process of PCR is repeated on each of the new DNA molecules
How is cycling made possible
the large excess of primers and dNTPs added at the beginning
What is special about the DNA polymerase used in PCR
it is thermal stable, meaning we do not need to add new DNA polymerase since it can survive the cyclical denaturation steps
If we did not use Taq polymerase, what would happen
the DNA polymerase would be denatured in the denaturing step at 95°C
What are the ingredients to PCR
Template DNA
DNA primers
dNTPs
DNA polymerase
Salts/Buffer
What is the purpose of template DNA in PCR
Source of DNA sequence that you seek to amplify
What is the purpose of DNA primers
Primers are short, single stranded DNA molecules called primers that base pair with the upper and lower strands of the region and flank the target sequence
What is the purpose of dNTPs
Nucleotide triphosphates are the raw ingredients for DNA replication
“N” stands for A,G, C,T
What is the purpose of DNA Taq polymerase
it is a heat tolerant polymerase from bacteria that catalyzes the formation of a new strand of DNA using dNTPs and the existing DNA strand as a template
What is the purpose of salts and buffer? What is specifically included in it
the buffer contains magnesium which DNA polymerases require
What are the typical temperatures for a thermocycler for PCR
1) 95 °C ( denature)
2) 55 °C (primer annealing)
3) 72 °C ( extension)
repeat cycle 30 times
What are the three requirements for a good DNA identity marker
Polymorphism
Equal Distribution
Detectable by PCR
What does polymorphic mean
it means that there must be many forms of the marker in the form of alleles
What is an example of a bad polymorphic DNA marker
ABO blood group gene, it is bad because it only has three different alleles
What does it mean to say that an allele must have roughly equal frequencies
the alleles must be evenly distributed across the population, for example if there are several alleles at a locus but only one of them is common, then it is not a good marker
Why is it important that a DNA identity marker is detectable by PCR
PCR amplifies the DNA
What are short tandem repeat (STR)s?
They are short repeats of DNA that are located at specific locations in the genome
Why are STRs good identity markers?
The number of repeats can vary among individuals
What is a tetramer
it is a 4 base pair sequence that repeats
what is it called when multiple loci are amplified simultaneously
multiplexing
what is the purpose of multiplexing? why can it be difficult to design
it saves time and money but the primers must have similar annealing temperatures
What is a Amelogenin sex-typing locus
it is a gene located on both the X and Y chromosomes but the X PCR product has an insert that makes it longer than the Y
what does it mean for an individual to be homozygous
it means they will have the same number of repeats on the chromosomes
what does it mean for an individual to be heterozygous
it means they have a different number of repeats on the chromosomes
What is the purpose of fluorescent tags
they are used in the detection of the PCR products as the amplicons carry tags that will fluoresce at different wavelengths
What is capillary gel electrophoresis
it is a form of PCR separation conducted in a thin tube
why is capillary gel electrophoresis better
it allows separations by 1 bp difference and it is faster
STR amplicons containing more repeats will be
longer
what is a electropherogram
it charts the wavelength and amplitude of the signal against the time since injection
why is an electropherogram used
it provides a unique genetic profile of an individual since no two electropherograms will be the same
what is the exception for an electropherogram
identical twins will have the same profile but fraternal will not
What are the factors that affect the annealing temperature
the GC/AT content
the length of the primer
primers with higher GC content will have what type of annealing temperature
they will have higher annealing temperatures due to the 3 hydrogen bonds
primers rich in AT content will have what type of annealing temperature
they will have lower annealing temperatures
longer primers will have ______ annealing temperatures in comparison the shorter primers
higher
