Exam 3 Flashcards
master
Fluorescent Dye functions
Fluorescent dye is attached to the TaqMan probe in quantitation step.
For amplification, dyes are attached to the primers
They can be used in multiplex PCR reactions
Singleplex PCR reaction
a single primer set used to amplify a single region (amplicon)
Multiplex PCR reaction
amplify over 20 loci at the same time
saves a large amount of time
can differentiate between individuals
How does having multiple dyes allow for multiplexing of PCR
It enables a clear differentiation of multiple targets, even when similar in size, by assigning fluorescence signals to each target, allowing for both simultaneous detection and better resolution in the analysis
How PCR products are labeled with fluorescent dyes for STR analysis
fluorescently labeled primers enable the differentiation of loci based on both size and dye color
How PCR products are labeled with a fluorescent dyes for mtDNA sequencing
fluorescently labeled ddNTPs allow for a sequential, base-by-base redout of the DNA sequence
How restriction endonucleases can be used to determine a person’s STR prodtfile
Restriction enzymes can be used to generate unique DNA fragment patterns based on STR length, but they lack specificity, sensitivity, and efficiency needed for reliable forensic STR profiling.
Basic function of restriction endonucleases
molecular “scissors” that enable precise cutting of DNA at specific sequences, playing a crucial role in genetic analysis, engineering, and forensic science
Structure of Variable Number Tandem Repeats (VNTRs)
consist of a sequence of DNA bases repeated back-to-back at a specific locus in the genome. Number of repeats can vary(genetic diversity)
Structure of Short Tandem Repeats (STRs)
subset of VNTRs where the repeating units are shorter, usually between 2-6 base pairs. vary in number of repeats, creating differences in length across individuals
Advantages of using PCR based assays in forensic investigation
high sensitivity
analysis of degraded samples
rapid and efficient process
ability to target specific regions
high throughput and multiplexing capability
compatibility with mixed and contaminated samples
standardization and databases
automation and portability
How PCR and capillary electrophoresis can be used to determine the number of repeats within an STR
by combining PCR to amplify STR regions and CE to separate and detect the amplified fragments, you can determine the number of repeats at each STR locus.
Advantages of using microsatellites over minisatellites
high sensitivity
efficiency
compatibility with degraded or small DNA samples
short length
ability to generate unique profiles
standardization in databases give an advantage
Basic information and characteristics for an STR to be usable
High variability
Stability and inheritance
Small size
low mutation rate
unique chromosomal locations
compatibility with PCR
STR types based on the number of base pairs in the repeat unit
Dinucleotide Repeats
Trinucleotide Repeats
Tetranucleotide Repeats
Pentanucleotide Repeats
Hexanucleotide Repeats
STR types based on Repeat pattern or sequence arrangment
Simple STR
Compound STR
Complex STR
STR based on Chromosomal Location
Autosomal STR
Y-STR
X-STR
Core loci
specific STR locations on the human genome that are routinely analyzed in forensic DNA testing.
why was core loci set expanded in 2017
Increased discrimination power
higher success rates with degraded samples
enhanced compatibility with international databases
improved kinship analysis
How can an internal lane standard be used to size the DNA fragments within an electropherogram peak
Calibration reference
Correction for variability
Fragment sizing
Allelic ladders and how they are used to “call” alleles for peaks
set of DNA fragments at a particular STR locus.
software visually compares the migration patterns of the sample peaks to the allelic ladder’s peaks
Steps in order to determine alleles, genotypes, and a profile
understanding the electropherogram
identify the loci
compare peaks to the allelic ladder
determine the alleles
determine the genotype
construct the DNA profile
Interpret results for autosomal STR data
Inclusion (match): sample could come from the individual in question
Exclusion (non-match): excludes the individual as the source
Inconclusive: if sample is degraded or has low quality
Interpret results for mtDNA data
Inclusion (match): cannot uniquely identify an individual
Exclusion (non-match): individual is excluded
Inconclusive
cannot exclude
Interpret results for Y-STR data interpretation
Inclusion (match): includes the individual
Exclusion (non-match): excluded as a source
Inconclusive
Cannot exclude
Identify all different factors impacting STR genotyping
Sample quality, PCR conditions, Allelic variation, technical limitations, human error, and genetic factors
How Tri-allelic patterns form
biological mechanisms, genetic abnormalities, or technical issues
How point mutations in the STR flanking region can impact primer binding
Failure to bind; STR locus will appear as a null allele
Reduced Binding Affinity; weaker PCR products
Mispriming; lead to false peaks or primer-dimer artifacts
How point mutations in the STR flanking region can impact Amplification
Incomplete Amplification; partial STR profile will be generated
Allelic Dropout; genotyping errors
How point mutations in the STR flanking region can impact STR profiling
Loss of information; difficult to distinguish between two individuals
False Genotyping; lead to wrong conclusions
How strutter products are formed
byproducts of DNA polymerase slippage during PCR amplification of STR regions.
shorter, smaller peaks, complicate interpretations of STR profiles
Basis of non-template adenylation
phenomenon where a polymerase (Taq polymerase) adds extra adenine residues to the 3’ ends of PCR products.
Non-template adenylation and its impact on samples
Size shifts, false alleles, artifact peaks, or allelic misinterpretations
Cause of Pull up peaks in electropherograms
signal from a fluorescent dye is used for one STR locus “pulls up” into the detection range of another adjacent peak from a different fluorescent dye or locus
spills or bleeds into adjacent channel
Cause of Spikes in Electropherograms
can arise from small technical issues with the capillary electrophoresis system
contaminants like dust, bubbles
electrical noise in the detection system
Identify a degraded DNA profile/ understand why it looks like that
characterized by shorter, fragmented STR products, missing alleles, and fainter peaks in the electropherogram
can be caused due to environmental factors, time, microbial activity, handling conditions, or mechanical damage
Complications associated with low copy number DNA testing and increasing cycle numbers
increased stochastic effects, PCR artifacts, non-specific amplification, and a greater risk of contamination
Characteristics and identifying mixture profile
multiple alleles at one locus, imbalanced peak intensities, allelic drop-out, missing or additional peaks, and complex patterns
careful interpretation is required when involved a low-quanitiy or degraded samples case
determine possible genotypes in a mixture sample
evaluate the number of peaks at each locus, their relative intensities, and their possible allele pairings
Characteristics of mtDNA
Inheritance
circular structure
size
coding and non-coding regions
high copy number
small number of genes
no recombination
mutability
absence of histones
General structure of the mtDNA genome
The two strands
-heavy and light strand
Control region
-Non-coding region and D-loop
Origin of Replication
-Origin of Heavy strand replication
-Origin of Light strand replication
Gene Organization
-Protein-coding genes
-tRNA genes
-rRNA genes
Hypervariable Regions
Length Polymorphisms
refer to variations in the length of a DNA sequence between individuals
STRs and VNTRs
Sequence Polymorphisms
refers to variations in the nucleotide sequence of a given region of DNA
SNPs and Indels
What information can be gained from mtDNA
maternal ancestry
degraded samples
family lineage
mothers side tracing
what information can be gained from Y-STR
Y chromosome
paternal lineage
tracing male family lines
paternal relatives
ancient male migration patterns
How the addition of ddNTPs is used to sequence regions of DNA
terminated DNA chain elongation at specific points=set of fragmented DNA sequences
Why sequence PCR reactions only include 1 primer instead of 2
because the sequencing reaction is designed to extend one strand of DNA in the 5’ to 3’ direction. starting point for DNA polymerase
Sequence Polymorphisms for mtDNA
Align the sequenced DNA to a reference
Identify differences such as SNPs, insertions, or deletions
report polymorphisms by position and type (transition, transversion, insertion, deletion)
put the findings in the context of population databases or haplogroups to provide insight into ancestry, lineage, or forensic identification
Heteroplasmy
Refers to the presence of more than one type of mitochondrial DNA (mtDNA) in a single cell or organism
How to report sequence heteroplasmy
Identify
document base change
quantify the proportion of each variant
contextualize the heteroplasmy
Y-STRs are all inherited together
they are located in close proximity on the Y chromosome and do not undergo recombination during meiosis
Exception to there only being one peak per locus and what causes it
homologous recombination or mutations that cause multiple alleles
exception occurs when there is a duplicated locus or mutations causing more than one peak at a given Y-STR marker
Rapidly mutating Y-STRs
class of Y-chromosome Short Tandem Repeats that exhibit a higher mutation rate compared to typical Y-STR markers
Rapidly mutating Y-STRs potential advantages
Increased resolution for kinship testing
forensic applications
Genealogical and Ancestry research
Personal Identification
