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Analyst Training > Amplification > Flashcards

Amplification Flashcards

1
Q

what is adenylation

A
  • 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
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2
Q

what are alleles

A

specific variation at a locus

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3
Q

what are amplicons

A
  • 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
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4
Q

what is an amplification positive control (PC)

A
  • 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
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5
Q

explain annealing

A
  • 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
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6
Q

explain the annealing temperature in relation to PCR efficiency

A

G-C rich primer sequences require higher temperatures (3 hydrogen bonds) than A-T sequences (2 hydrogen bonds)

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7
Q

what is an amplification negative control (AB)

A
  • assesses contamination of PCR reagents or consumables
  • contains all reagents added to the PCR reaction except for template DNA (replaced by suspension buffer)
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8
Q

what are base pairs

A
  • tetra-nucleotide base pairs are the most common (ex: GATA)
  • stutter percentage increases as the number of repeating base pairs decreases
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9
Q

what is bovine serum albumin (BSA)

A
  • protein added to help stabilize polymerase
  • reduces/prevents PCR inhibition
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10
Q

what is buffer tris-hcl

A
  • provides a stable pH environment
  • incorrect pH can alter the quality of dNTP insertions
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11
Q

what are some challenges of STR amplification

A
  • input DNA
  • stochastic effects
  • PCR inhibition
  • degradation
  • stutter
  • incomplete non-template addition
  • primer binding site mutation
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12
Q

what are complex hypervariable repeats

A
  • 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
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13
Q

what are complex repeats

A
  • 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
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14
Q

what are compound repeats

A
  • 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
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15
Q

what is degradation in relation to STR challenges

A

environmental conditionals cause DNA to break into smaller pieces

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16
Q

what is denaturation

A
  • 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
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17
Q

what are dinucleotide triphosphates (DNTPs)

A
  • 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
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18
Q

what is dye labeling

A
  • 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
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19
Q

what is extension

A
  • 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
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20
Q

what are some factors affecting PCR efficiency

A
  • 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
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21
Q

what is final extension

A
  • 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
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22
Q

what is an incomplete non-template addition in relation to STR challenges

A
  • failure of adenylation to occur
  • one base pair shorter than expected (minus A)
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23
Q

define inhibitors in relation to PCR efficiency

A

presence of inhibitors will affect the efficiency of the PCR reaction

24
Q

explain inhibitors in relation to STR challenges

A
  • 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
25
Q

what is input DNA in relation to STR challenges

A
  • 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
26
Q

what is length variation

A
  • short tandem repeats (STRs)
  • variable number tandem repeats (VNTRs)
  • detected with the analysis methods for micro and mini satellites
27
Q

what is a locus/loci

A

the area on an DNA sequence where repeat patterns are located

28
Q

what does magnesium chloride (MgCl2) do

A
  • 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
29
Q

what are master mixes

A
  • contains all components of a process except for DNA
  • use overage to compensate for slight pipetting differences
30
Q

what are some methods for dye labeling

A
  • intercalating dyes (post-PCR)
  • dye-labeled nucleotide insertion (during PCR)
  • dye-labeled primer insertion (during PCR)
31
Q

explain the number of cycles in relation to PCR efficiency

A
  • 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
32
Q

what is an oligonucleotide

A
  • short DNA or RNA molecules
  • contain a small number of nucleotide base pairs
33
Q

what are the PCR components and what do they do

A
  • 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
34
Q

what is potassium chloride (KCl)

A
  • 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
35
Q

what are the components of the PowerPlex Fusion 6C kit

A
  • 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)
36
Q

what are primers

A
  • 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
37
Q

what are primers in relation to PCR efficiency

A
  • mportant to pick primers that don’t form primer-dimers
    • primer-dimers anneal to themselves instead of the target, decreasing the amp product
38
Q

what is a primer binding site mutation in relation to STR challenges

A
  • 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
39
Q

explain quality of DNA in relation to PCR efficiency

A

degraded DNA will affect the results obtained

40
Q

what do reaction components mean in relation to PCR efficiency

A
  • 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
41
Q

what is a reaction pH in relation to PCR efficiency

A
  • influences the stability of the primers and reaction activities
  • if not regulated properly, the pH could result in a decrease in amp product
42
Q

what is a sequence variation

A

a type of DNA marker variation that is detected with SNP and insertion/deletion analysis methods

43
Q

what are short tandem repeats (STRs)

A
  • 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
44
Q

what are simple repeats

A
  • 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
45
Q

what are single ncleotide polymorphisms (SNPs)

A
  • genomic variant at a single base pair
  • ex: GATA vs. GATC
46
Q

what are stochastic effects in relation to STR challenges

A
  • 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
47
Q

what is STR amplification

A
  • targets specific regions of DNA
  • detects STR fragments during capillary electrophoresis
  • purpose is to generate millions of copies of target regions
48
Q

what are STR markers

A
  • short DNA sequences that are repeated several times in a row
  • highly repetitive and amplified using PCR
49
Q

what is stutter in relation to STR challenges

A
  • 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
50
Q

explain target fragment length in relation to PCR efficiency

A
  • shorter regions amplify more efficiently
  • larger regions are more affected by degradation
51
Q

what is taq polymerase and what does it do

A
  • 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
52
Q

what is template DNA

A
  • 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
53
Q

what is the purpose of thawing

A
  • ensures frozen DNA extracts and reagents are room temperature
  • need a homogenous mixture so the concentration is the same
54
Q

what is a thermal cycler

A

rapidly heats and cools DNA at consistent temperatures for PCR

55
Q

explain thermal cycler performance in relation to PCR efficiency

A

wrong program, wrong parameters, or cycling malfunctions will affect the final PCR product

56
Q

what are variable number tandem repeats (VNTRs)

A
  • 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
57
Q

what is vortexing

A

ensures a homogenous mixture and prevents DNA from adhering to the sides of the tubes

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