Amplification Flashcards
what is adenylation
- a type of non-template addition that occurs at the end of extension
- an adenine is added to the end of a DNA fragment by taq polymerase
what are alleles
specific variation at a locus
what are amplicons
- PCR products are referred to as amplicons
- beginning at the third PCR cycle, amplicons are doubled per cycle (exponential)
- they lose efficiency at higher cycle numbers
- amplicons are generated as a result of primer hybridization during CE
what is an amplification positive control (PC)
- demonstrates that amplification was successful
- is included in STR typing kits and contains known genotypes
- unexpected results indicate improper pre, reagent failure, or incorrect thermal cycle
explain annealing
- second step of PCR
- lowers the temperature to allow the various primers anneal to their targets on the separate DNA strands
- polymerase attaches to the primer-DNA bond to start copying the template
- the higher the annealing temperature, the more specific the binding
explain the annealing temperature in relation to PCR efficiency
G-C rich primer sequences require higher temperatures (3 hydrogen bonds) than A-T sequences (2 hydrogen bonds)
what is an amplification negative control (AB)
- assesses contamination of PCR reagents or consumables
- contains all reagents added to the PCR reaction except for template DNA (replaced by suspension buffer)
what are base pairs
- tetra-nucleotide base pairs are the most common (ex: GATA)
- stutter percentage increases as the number of repeating base pairs decreases
what is bovine serum albumin (BSA)
- protein added to help stabilize polymerase
- reduces/prevents PCR inhibition
what is buffer tris-hcl
- provides a stable pH environment
- incorrect pH can alter the quality of dNTP insertions
what are some challenges of STR amplification
- input DNA
- stochastic effects
- PCR inhibition
- degradation
- stutter
- incomplete non-template addition
- primer binding site mutation
what are complex hypervariable repeats
- have numerous non-consensus alleles that differ in size and sequence
- ex: GATA GATA GATA GGGGGG CGTA CATA AGCA
- core CODIS loci that are complex hypervariable repeat STR markers:
- SE33
what are complex repeats
- contains repeats of variable length as well as variable sequences
- ex: GATA GATA GATA GTA GATA GTAA GATA
- core CODIS loci that are complex repeat STR markers:
- D21S11
what are compound repeats
- consists of repeating sequences of identical length but a variable sequence
- ex: GATA GATC GATT
- core CODIS loci that are compound repeat STR markers:
- VWA, FGA, D3S1358, D8S1179
what is degradation in relation to STR challenges
environmental conditionals cause DNA to break into smaller pieces
what is denaturation
- first step of PCR
- disrupts the hydrogen bonds of double-stranded DNA
- raises the temperature to be able to denature and separate the two strands of DNA
- because of the high temperature, all enzymatic reactions stop
- incomplete denaturation allows the separated strands to “snap back” together
what are dinucleotide triphosphates (DNTPs)
- used to create a replicate DNA strand
- Adenine, Cytosine, Guanine, and Thymine
- a nucleotide consists of a base(A, C, T, G) bonded to a sugar, bonded to a phosphate group
- added to PCR in equal concentrations for optimal incorporation
- lower concentrations of dNTPs minimize mispairing and reduce the likelihood of extending miss-incorporated nucleotides
- miss-incorporated bases cannot be proofread since Taq polymerase lacks the 3’ to 5’ exonuclease activity; promotes chain termination
what is dye labeling
- dye labeling primers is the most common method for forensic STR DNA analysis
- uses a fluorophore, a molecule capable of fluorescing upon excitation
- the dyes are attached to one primer at the 5’ end in a pair
- the labeled primers are incorporated during amplification, and the color of its fluorescence is detected during CE
- only one primer is labeled because the forward and reverse primers have different sequences and can affect migration differently
what is extension
- third/last step of PCR
- raises the temperature so that DNA (taq) polymerase can activate the primer’s fluoresce
- continues to add complementary nucleotides to the single-stranded DNA
what are some factors affecting PCR efficiency
- annealing temperature
- choice of primers
- concentration of reaction componenets
- number of cycles
- presence of inhibitors
- quality of DNA
- reaction pH
- target fragment length
- thermral cycler performance
what is final extension
- occurs to ensure complete adenylation
- without adenine overhang, the egram will show (-A) peaks next to the parent peaks
- a final, 10-minute extension removes the -A peaks
what is an incomplete non-template addition in relation to STR challenges
- failure of adenylation to occur
- one base pair shorter than expected (minus A)
define inhibitors in relation to PCR efficiency
presence of inhibitors will affect the efficiency of the PCR reaction
explain inhibitors in relation to STR challenges
- when a substance is present that prevents proper amplification
- renders PCR components inactive and may result in preferential amp, complete amp failure, or partial profiles
- present in samples from crime scenes or biological fluids
what is input DNA in relation to STR challenges
- input DNA goal in 1ng
- too little DNA cause cause stochastic effects
- too much DNA can cause non-specific binding, artifacts, preferential amplification, and inhibition
what is length variation
- short tandem repeats (STRs)
- variable number tandem repeats (VNTRs)
- detected with the analysis methods for micro and mini satellites
what is a locus/loci
the area on an DNA sequence where repeat patterns are located
what does magnesium chloride (MgCl2) do
- required by DNA polymerase to function
- the concentration of magnesium chloride may affect:
- annealing
- strand dissociation temperatures
- product specificity
- formation of artifacts
- enzyme activity
- increasing concentration increases yield, but decreases specificity and fidelity
what are master mixes
- contains all components of a process except for DNA
- use overage to compensate for slight pipetting differences
what are some methods for dye labeling
- intercalating dyes (post-PCR)
- dye-labeled nucleotide insertion (during PCR)
- dye-labeled primer insertion (during PCR)
explain the number of cycles in relation to PCR efficiency
- PCR efficiency decreases with more cycles - reagents and samples run out
- taq polymerase is rendered inactive after adding a certain number of bases
- a higher number of amplicons increases the re-annealing, reducing the chance of primer annealing
what is an oligonucleotide
- short DNA or RNA molecules
- contain a small number of nucleotide base pairs
what are the PCR components and what do they do
- Bovine Serum Albumin (BSA) to stabilize Taq polymerase
- buffer to provide a stable pH environment for the dNTPs
- DNA polymerase (Taq polymerase) to help with replication and activate fluorescence
- dNTPs to help with replication
- magnesium chloride to help polymerase function
- potassium chloride to neutralize the negative charge of DNA
- primers to anneal to specific regions of the DNA
- template/sample DNA
what is potassium chloride (KCl)
- neutralizes the charge present on the backbone of DNA
- facilitates annealing of the primer to the template DNA
- reduces the “repulsion” between negatively charged DNA strands
what are the components of the PowerPlex Fusion 6C kit
- 5X Master Mix (pre-amp)
- 5X Primer Mix (pre-amp)
- 2800M Control DNA 10ng (pre-amp)
- amplification grade water (pre-amp)
- allelic ladder (post-amp)
- WEN ILS 500 (post-amp)
what are primers
- small oligonucleotides that anneal to complementary strands of a specific region of DNA
- typically 15-25 bases long
- forward and reverse primers are required for an exponential rate of amplification
- PCR includes amplification, degradation, IPC, and Y-specific primers
what are primers in relation to PCR efficiency
- mportant to pick primers that don’t form primer-dimers
- primer-dimers anneal to themselves instead of the target, decreasing the amp product
what is a primer binding site mutation in relation to STR challenges
- single base mutation in site
- can have no effect, or can cause imbalance or dropout
- degenerate primers are added to known primer binding site mutations
explain quality of DNA in relation to PCR efficiency
degraded DNA will affect the results obtained
what do reaction components mean in relation to PCR efficiency
- too little reagents/components, then the reaction stops too soon
- too much, then the reagents/components inhibit or decrease specificity
- excess of primers and Mg promotes non-specific binding
what is a reaction pH in relation to PCR efficiency
- influences the stability of the primers and reaction activities
- if not regulated properly, the pH could result in a decrease in amp product
what is a sequence variation
a type of DNA marker variation that is detected with SNP and insertion/deletion analysis methods
what are short tandem repeats (STRs)
- microsatellite; detects length variation
- currently used in human identity testing
- a repetitive unit of 1-6 base pairs
- a 4 base pair unit is the most common (tetra-nucleotide)
- ex: GATA
what are simple repeats
- a type of STR repeat that consists of one repeating sequence of identical length
- ex: GATA GATA GATA GATA
- core CODIS loci that are simple repeat STR markers:
- TOPX, CSF1PO, D5S818, D13S317, D16S539
- some simple repeats are microvariants; don’t contain full identical repeats
- ex: GATA GATA GATA GAT__
- core CODIS loci that are simple repeat microvariant STR markers:
- TH01, D18S51, D7S820
what are single ncleotide polymorphisms (SNPs)
- genomic variant at a single base pair
- ex: GATA vs. GATC
what are stochastic effects in relation to STR challenges
- unbalanced amp of heterozygote loci due to low DNA input
- can result in peak imbalance, false homozygosity, total dropout, or different alleles being detected during subsequent amplifications
what is STR amplification
- targets specific regions of DNA
- detects STR fragments during capillary electrophoresis
- purpose is to generate millions of copies of target regions
what are STR markers
- short DNA sequences that are repeated several times in a row
- highly repetitive and amplified using PCR
what is stutter in relation to STR challenges
- reproducible, known, and expected amplification artifacts
- taq polymerase because inactive after a certain number of bases, detaching from the DNA
- active enzymes will attach instead, but don’t align the DNA properly
- strand slippage is when a fragment is one repeat shorter than the expected size
explain target fragment length in relation to PCR efficiency
- shorter regions amplify more efficiently
- larger regions are more affected by degradation
what is taq polymerase and what does it do
- DNA polymerase adds complimentary dNTPs to the single-stranded DNA
- taq polymerase is stable at high temperatures, which are required to denature the DNA into two strands
- annealing at regular temperatures creates non-specific products
- Hot Start Taq is chemically modified to require heat activation before polymerase activity begins
- only moves in the 5’ to 3’ direction
- tends to add a single adenine to the end of a DNA fragment, creating an overhang
- this is called adenylation
what is template DNA
- needs to be a specific concentration for amplification to work properly
- target = 1 ng DNA
- too much will create more artifacts in the egram
- too little with give poor egram yields
what is the purpose of thawing
- ensures frozen DNA extracts and reagents are room temperature
- need a homogenous mixture so the concentration is the same
what is a thermal cycler
rapidly heats and cools DNA at consistent temperatures for PCR
explain thermal cycler performance in relation to PCR efficiency
wrong program, wrong parameters, or cycling malfunctions will affect the final PCR product
what are variable number tandem repeats (VNTRs)
- minisatellite; used to detect length variation
- a repetitive unit of 20-100 base pairs
- longer repeats than STRs, and more variation of the amount of base pairs in the repeat
what is vortexing
ensures a homogenous mixture and prevents DNA from adhering to the sides of the tubes
