gel electrophoresis,
A match or not a match: That is the question ...
1. Inclusion (match): Peaks between the compared STR profiles have the same genotypes and no unexplained differences exist between the samples. Statistical evaluation of the significance of the match is usually cited in the match report. 2. Exclusion (nonmatch): The genotype comparison shows profile differ- ences that can only be explained by the two samples originating from different sources. 3. Inconclusive: The data does not support a conclusion as to whether the profiles match. This finding might be reported if two analysts remain in disagreement after review and discussion of the data and it is felt that insufficient information exists to support any conclusion.
irisplex assay
6 snp's multiplex assay her2 gene, rs12913832 -main gene related to eye color OCA2 gene, rs1800407 SLC24A4 gene, rs12896399 SLC45A2 (MATP) gene, rs16891982 TYR gene, rs1393350 IRF4 gene, rs12203592 extraction -QIAMP DNA mini kit quantiifcation -quantifiler trio amplification -2 step ptocess -pcr extension, and single base-pair extensions (SBE) PCR -fluorscently labeled nucelotides instead of fluorscently labeled primers like STR profile PCR -poly T tails were also added to the single base pairs extenion primers to inc. detection and seperation of the pcr products capillary electorophoresis -applied biosystems peaks in differen dye channels--> diff. alleles at each snp location
multiple amplification sets
After the primers are chemically synthesized, they are combined to form multiplex amplication sets. For PCR amplication followed by fluorescence detection, each STR marker requires two primers, one that is labeled with a fuorescent dye and one that is not. Different fluorescent dyes are used in order to increase the level of multiplexing possible with multi-color fluorescence detection Because the two primers define the end of the PCR product, oligonucleotide synthesis failures can impact the length of the subsequent amplicon.
Peak sizing with DNA fragment analysis.
An internal size standard, such as GS500-ROX, is analyzed along with the DNA sample and used to calibrate the peak data points to their DNA size. This standard is labeled with a different color fluorescent dye, in this case ROX (detected as red), so that it can be spectrally distinguished from the STR alleles that are labeled in other colors.
Off-ladder alleles can be verified by rerunning the amplified product, reamplifying the sample, or by amplifying the sample with single-locus primers.
Heterozygous samples with one 'normal' allele and one microvariant allele make it easy to confirm the microvariant. the normal allele with a full-length repeat sequence will fall in an allele bin from the allelic ladder, while the microvariant allele possessing a partial repeat sequence will fall between the allele bins created by the allelic ladder.
Partial STR profiles
If the genomic DNA in a sample is severely degraded or PCR inhibitors are present, only a partial STR profile may be obtained. Usually the larger STR loci in a multiplex reaction, such as D18S51 and FGA, will be the first to fail on a degraded DNA sample. the significance of a match will decrease because there are fewer loci to compare.
Gel vs. Capillary Electrophoresis
In capillary electrophroesis there a few vital components used. First off there is the capillary, through which the DNA sample flows during separation. Each end of the capillary is positioned in a reservoir containing an electrolyte (buffer solution). The source reservoir contains a positive electrode and the destination reservoir contains a negative electrode. At the source reservoir there is a sample vial that will be used at the start of the process in order to load the DNA sample into the source end of the capillary. Both the anode and cathode are connected to a high-voltage power supply. Lastly, near the end of the capillary is a detector, which is connected to a computer for data analysis. The DNA sample is loaded into the source end of the capillary by electrokinetic injection or through a vaccum. When the power supply is turned on, it provides charge to the anode and cathode creating an electric field between the reservoirs. This initiates the migration of both positively and negatively charged ions in the DNA sample by electroosmotic flow. Further, the detector detects the ions near the end of the capillary, which have separated during their migration according to their charge, size, and shape. Note that differences in charge and size causes the ions to move at different speeds. The molecules are detected close to the end of the capillary, usually by ultraviolet absorption. The data is then observed in electropherograms, which display the detector output against time. Under gel electrophoresis, it involves running a current through a gel containing samples of interest (in this case DNA molecules). The gel used in this process is typically an agarose gel. Before the DNA samples are added the gel is placed in the gel box. On each side of the box there is a negative electrode and a postive electrode. Inside the box where the gel is placed, a buffer is added to each chamber. The buffer is full of solutes that can conduct current forming a uniform distribution of ions from one electrode to the other. Once the gel is completely submerged, the DNA samples are transfered into the wells carefully; one well consists of the DNA ladder used as a standard reference. Once the power to the gel box is turned on, current starts to flow through the gel. DNA molecules (negatively charged) become mobilized and start moving towards the positve electrode. As the gel runs, the shorter DNA fragments travel through the pores of the gel matrix faster than the larger DNA fragments. This is due to the fact smaller DNA fragments collide less with the agarose allowing it to move through the gel more quickly. As a result, the shorter pieces of DNA will be closer to the positve electrode end of the agarose and the larger pieces of DNA will remain near the wells. Further, a tracking dye is used to serve as a visual indicator for when to stop the electrophoresis process. Once the dye front reaches the end of the gel the power is cut off and the gel is ready to be examined.
Dye blobs occur when fluorescent dyes come off of their respective primers and migrate independently through the capillary.
Most dye artifacts, which are created through incomplete attachment of the fluorescent dye during oligo- nucleotide (primer) synthesis, are typically removed or significantly reduced through primer purification performed by the STR kit manufacturer.
During a run, the system:
Prepares the capillary by pumping fresh polymer solution under high pressure from the polymer delivery pump to the waste position in the Cathode Buffer Container (CBC). Electrokinetically injects the sample into the capillary using a low-voltage for a few seconds. Washes the capillary tips in the rinse position of the CBC, then returns the capillary to the buffer position of the CBC. • Ramps the voltage up to a constant voltage. -A high electric field is created between the ground end of the Anode Buffer Container (ABC) and the negative voltage applied to the load header of the capillary array. -This field pulls the negatively charged DNA through the separation polymer. The smaller fragments migrate faster than the larger fragments and reach the detector first. -To insure optimal separation and maintain denaturation of the DNA, the capillaries are thermally controlled in the oven and in the detection cell. The oven has a Peltier heat unit and fan-circulated air. The Peltier can heat and cool the oven to maintain sub-ambient temperatures, which are useful for non- denaturing applications such as SSCP (Single-strand conformation polymorphism). • In the detection cell, the dyes attached to DNA are excited by a narrow beam of laser light. The laser light is directed into the plane of the capillaries from both the bottom and top. A small amount of laser light is absorbed by the dyes and emitted as longer wavelength light in all directions. • Captures the fluorescent light on the instrument optics while blocking the laser light. The light passes through a transmission grating, which spreads the light out. The light is imaged onto a cooled, scientific-grade CCD array. For each capillary, 20 zones on the CCD are collected to provide20-color data for each capillary. • Converts the 20-color data into multi-dye data for the entire run. For sequencing applications, 4 different dyes are used to determine the 4 bases A, G, C and T. For fragment and HID analysis applications, up to 6 dyes can be used in a single run for higher throughput.
Incomplete 3'(+A) nucleotide addition results with amplifications containing too much DNA template or thermal cycling conditions that affect the optimization of the PCR reaction.
The Taq DNA polymerase used for amplifying STR loci will catalyze the addition of an extra nucleotide, usually an 'A' (adenine), on the 3 end of double-stranded PCR products. when incomplete 3' nucleotide addition occurs, 'split peaks' will result sometimes referred to as +/- A, or N and N+ 1 peaks. In these cases, the allele of interest will be represented by two peaks 1 bp apart
The allelic ladder is the standard to which STR alleles are compared to obtain the sample genotype.
The alleles in an allelic ladder need to be resolved from one another and above the peak detection threshold of the data collection and analysis software in order to correctly call STR alleles in unknown samples. The sizes obtained for each allele in the allelic ladder are used to make the final genotype determination in the unknown samples.
The HPLC separation results for four different multiplex STR amplification kits
The combination of reverse-phase and ion-exchange properties in the HPLC column aid the separation characteristics. Unlabeled primers elute from the column first. The fluorescent dye label attached to one primer in each locus-specific pair was observed to impact the oligonucleotide retention on the HPLC column. This can best be seen in the kit B primer set separation. The nine peaks in the time range of 9-15 min are unlabeled oligonucleotides. Peaks 10-17, which pass the detector between 19 and 26 min, are either JOE or 5-FAM dye labeled. Finally, peaks 18-20 are NED- labeled. Thus, the properties of the NED dye are such that oligonucleotides labeled with this dye are retained longer on the HPLC column than 5-FAM and JOE labeled primers. To further confirm which peaks were labeled with fluorescent dyes, a single wavelength fluorescent detector was used in conjunction with the HPLC separation.
Multiplex STR primer sets can be subjected to a rapid quality control check by direct analysis with TOF-MS.
The presence or absence of a peak at a particular mass can then be used to verify that a particular primer is present in the mix
results
The software generates an electropherogram (intensity plot) for each dye based on the migration of DNA fragments over the run and generates primary analysis results: For sequencing applications, the electropherogram is adjusted to compensate for slight mobility differences due to the dyes, then basecalling is performed and quality values are assigned. For fragment and HID analysis, the software uses the internal size standard to assign a fragment size and a sizing quality value to each peak. If the autoanalysis functionality has been set up, the system transfers the sample data to a secondary analysis software application for further processing. Alternatively, you can manually transfer the sample data to a secondary analysis software application for further processing.
Epigenetics
The study of changes in an organisms phenotypes cause by modification to the expression of an organism's genotype rather than changes to the genetic code itself
Genotyping is performed through a comparison of sized peaks from PCR-amplified samples to allele size bins.
These allele bins are defined with the genotyping software using size information from an allelic ladder run with each batch of samples. Any peak falling in a particular dye color and allele bin size range is designated as an allele for that locus. Peaks in both the allelic ladder and the PCR-amplified samples are sized using the same internal size standard so that they may be compared to one another. A common size range for the genotyping allele bins is 0.5bp around each allele. This size range enables PCR products that are 1bp different from one another to be differentiated; Any peak with the same dye-color label that is sized within half a base pair of an allelic ladder is designated by the STR genotyping software as being that allele.
Occasionally a sample may contain an allele that does not fall within 0.5 bp of an allele from the corresponding locus- specific allelic ladder.
These alleles are designated as 'off- ladder' alleles or variant alleles. The off-ladder allele peak may be larger or smaller than the alleles spanning the allelic ladder range or it may fall in between the rungs of the allelic ladder.
Triallelic patterns result from extra chromosomal fragments being present in a sample or the DNA sequence where the primers anneal being duplicated on one of the chromosomes.
These rare anomalies are detected by an extra peak at a single locus The three peaks can either be of approximate equal intensity or possess peak heights such that two of the peaks sum up to approximately the height of the third allele
Preparing samples
When DNA samples are prepared for sequencing, fragment analysis, or HID analysis on the 3500 or 3500xL analyzer, fluorescent dyes are attached to the DNA. For most applications, the sample is denatured so that only single-strand DNA is present.
Stutter products are the most common source of additional peaks in an electropherogram of an STR sample.
When STR loci are PCR-amplified, a minor product peak four bases (n-4) shorter than the corresponding main allele peak is commonly observed in tetranucleotide repeats
gel electrophoresis part 2 process
agarose gel placed into the tank; used as a medium to separate molecules because its porous spongy matrix running buffer is added to the tank by pouring half of the buffer into one side of the chamber until the air trapped under the tray is pushed out remaining buffer is poured into the other side of the buffer gel should be fully submerged -buffer is full of solutes, conductivity creates a uniform distribution of ions from one electrode to the other electric charge flows through the gel, dna is mobilized and migrates to the positive electrode low intensity blue light switched on; can see wells in gel to load our dna samples samples are placed in wells; this particular gel has 6 wells because we have 4 samples samples are added to the wells with a micropipette; tip only placed half way into gel to avoid penetrating the bottom record order DNA samples are loaded and change pipette tips between each sample to avoid cross-contamination lid is replaced and unit is switched on current is on; it flows form the negative electrode to the positive electrode the smaller the molecule the less it will collide with the arose allowing it to move thorough the gel more quickly; smaller molecules are faster tracking dye serves as a visual indicator for when to stop the electrophoresis process; once the dye front reaches the end of the gel the power is cut off and the gel is ready to be examined each of the bands represents millions of DNA of similar size that are stuck within that section of the agarose
Mixtures of DNA from two or more individuals are common
an analyst must decide whether the source of the DNA in the questioned sample is from a single individual or more than one person. This may be accomplished by examination of the number of alleles detected at each locus as well as severely imbalanced peak height ratios or pronounced peaks in the stutter position
Peak detection thresholds
are set on capillary electrophoresis instruments below which any data is considered unreliable. A common peak detection threshold is 50 relative fluorescence units (RFUs). Only peaks above this user-defined analytical threshold are considered an analytical signal and thus recorded by the data analysis software. stochastic threshold, a point above which there is a low probability that the second allele in a truly heterozygous sample has not dropped out. If all observed peaks at an STR locus are above the interpretation threshold, then there is confidence that all amplified alleles are being detected.
how do you detect methylation?
bisulfite conversion unmethylated cytosine= uracil methylated cytosines--> unaffected and remain stable because of methylation identification of methylated cytosine
Air bubbles, urea crystals, or voltage spikes
can give rise to a false peak in an ABI 310 or other CE instrument. These peaks are usually sharp and appear equally intense throughout all four colors
peaks on electropherogram
correspond to the various STR alleles amplified from the DNA sample. present at various locations in a sample's electropherogram and are usually plotted as fluorescent signal intensity versus time passing the detector on a capillary electrophoresis system (or position on a gel once electrophoresis has been halted and the gel scanned)
steps for converting those fluorescent peaks into a descriptive number known as an 'allele call'
data collection--> color separation--> peak identification--> peak sizing--> comparison to allelic ladder--> genotype assignment to alleles--> data review--> confirmation results by second examiner
Short tandem repeat STR) markers have become wide- spread in their use by the forensic DNA typing community
due to their ability to provide a high degree of discrimination between individuals in a relatively short period of time and in spite of highly degraded sample material Multiplex STR analysis has improved the power of discrimination with the ability to analyze multiple regions of DNA simultaneously. From a single amplication with less than a nanogram of DNA template, it is possible to obtain random match probabilities of >1 in 100 billion
hirisplex assay
expansion of the Irisplex assay -24 dna variants -23 snp's and one insertion/deletion gene (INDEL) --6 snp's within the validatied irisplex assay --21 snps and one INDEL related to hair color prediction ----4 snps overlap
what is snp information for
forensic dna phenotyping -predicting human appearance from crime scene. material or investigation purposes coding regions of gene linked to externally visible characterastics (EVCs)
hirisplex classifications and prediction accuracy
hair color -blonde- 81% -brown- 75% -red- 92% -black- 85% shade -light -dark eyes -blue- 94% intermediate- 74% -brown- 95%
The internal size standard is necessary for proper sizing of DNA fragment peaks detected in an electropherogram
if any of the peaks in the size standard are below the peak detection threshold established in the data collection and analysis software, then the sizing algorithms will not work properly and STR alleles may be sized incorrectly. As part of data quality assurance, an analyst checks to make sure that the internal size standard peaks were all detected properly before proceeding to genotype the STR alleles in a sample.
primer design for pcr
in order to isolate DNA regions we are interest in we need to design primers -we design one forward primer and one reverse primer DNA is written in the 5' to 3' and the complementary is written 3' to 5' synthesis needs to originate from he 3' end and move in the 3' prime direction -design a forward primer it would need to bind onto the complementary strand of DNA 20 bp long you want it to anneal strongly especially on the ends (5') -if you ended the strand with a T it only has two bonds between the A and T -make it 21 bp and incorporate the C; anchors down the position when DNAP binds in once forward primer is complete the next step is to design the reverse primer -start at the 3' end and make it 20 bp long -ends on a G or C, good to keep it 20 bp long -must create reverse complement of this reverse strand of DNA so write the sequence at the 5' end CG content -20 bp primer with low GC bonds will have a lower annealing temperature then a primer with a higher GC content -low annealing temp means when temp. is increased in PCR this primer cannot bind on to our gene of interest and won't work -primers must be around 50% primers under 60 bp are in a certain price category, anything above the synthesize becomes less reliable sticky end example -are not complementary to original plasmid but instead line up with plasmid used with restriction enzyme on I want to insert this gene into -primer standard design is 20 bp on your gene of interest and 40 bp you are going to insert the gene of interest into
'pull-up,'
is a result of the inability of the detection instrument to properly resolve the dye colors used to label STR amplicons. This phenomenon is due to spectral overlap. A peak of another color is 'pulled up' or 'bleeds through' as a result of exceeding the linear range of detection for the instru- ment (i.e., sample overloading).
The Applied Biosystems 3500/3500xL Genetic Analyzers
is an automated 8 and/or 24 capillary instrument designed for a wide range of sequencing and fragment analysis applications. fully automated, from sample loading to primary data analysis, for sequencing, fragment analysis, and HID (human identification) analysis.
The matrix file (sometimes termed a spectral calibration)
is critical for proper color separation in an electropherogram. If the observed peaks are not associated with the proper dye label, then the sample genotype cannot be correctly determined. matrix files are established by running samples that contain each of the dyes individually. The results of the individual dye runs are combined to form a mathematical matrix that is used to subtract the contribution of other colors in the overlapping spectra.
short tandem repeats (STRs)
length based polymorphism that are utilized non-coding regions -mutate more rapidly and contain the largest portion of DNA variations
gel electrophoresis part 1
method of using an electric field to separate molecules based on their size and polarity dna is a negative charged molecule that can be cleaved into diff. sized fragments using restriction enzymes monitor run alls you to review the progress of a run in real time and gives you access to quarry control parameters direct viewing of base called files for sequencing experiments or size called files or fragment called runs
dna methylation
methyl group addition to cytosines -deactivates or activates gene expression -tissue specific -can be altered over time --- environmental factors chomatin -regulates access to dna transcription nucleosomes and histones -heterchomatin vs euchomatin -open--> expressed closed- not expressed
Age Estimation
popular fast expanding topic of research -benefits of establishing the age of trace dna samples --paired with trait prediction of forensic dna typing methylation profiling -dna methylation has a well established role in aging -terrible childhood/different stressors--> phsycially age prematurely compared to those who have not experienced such stressors
The Local Southern method works very well for accurate sizing of DNA fragments over the 100- to 450-bp size range necessary for STR alleles—
primarily because in this size range DNA molecules migrate during electrophoresis with an approximately linear relationship between size and separation time. signal intensity for any of the calibration peaks in the internal sizing standard is too weak, then unknown peaks in that region will not be sized accurately.
Applied bio systems 3500 series genetic analyzer for dna sequencing and fragment sizing
primary consumables including polymer, cathode, and anode buffers, and easy to install capillary array makes setting up the instrument fast and simple dispose of consumables properly after use all consumables contain novel integrated radio frequency identification or RFID tag labels; enable viewing tracking and reporting RFID tracking feature eliminates administrative tracking and reduces the chances of error
parabon snapshot
private company -face prediction based on genetic information -genetic genealogy -dna phenotyping -ancestry and kinship analysis expensive -4000-5000 per sample -unpublished source code
resulting electorpherograms
sensitive down to as little as 31 pg of DNA -multinomial logistic regression model -phenotypic prediction accuracy blue 91% intermediate- 73% brown- 93%
single nucleotide polymorphism (SNP)
sequence based polymorphisms occur during coding regions of DNA -single point mutations that are the diff. of a single nucleotide of a gene sequence
STR and DNA profiling
short tandem repeats composed of 2-8 nucleotides, located in non-coding region of chromosomes highly polymorphic ex.) inherit two different alleles, one from his mother and one from his father= heterozygous PCR is used to amplify specific regions of DNA, primers are designed to bracket PCR, and one primer of the pair is fluorescently labeled -4 fluorescent dyes are used amplified DNA fragments are allocated into the gel, fragment is detected when it passed by a laser near the bottom of the gel -the laser reads all four colors and displays and output of the PCR according to there size into fluorescent colors of amplified pieces each peak represents an allele and each locus occurs as a pair of peaks= heterozygous, or one peak= homozygous random match probability= specific STR profile icing randomly in a population -calculated using frequencies at which different str alleles occur in a population - p^2 + 2pq + q^2=1 -p is the freq. of the appearance of the A allele -q is the freq. of the appearance of the a allele -mathematical way of saying that in a population of alleles of a gene exists in a. state of equilibrium - multiply the frequencies together at each STR locus; STR1 STR2 x STR3....
hirisplex- S
third assay expansion -two dual multiplex assays -41 total snps --17 sips for strictly skin tone --24 previous snps for hair and eye color, 19 of which also contribute to skin tone 5 classification categories -very pale- 75% -pale- 73% - intermediate- 75% - dark-84% - dark to black- 98%
predicting EVC's of an individual
using genotypic information -coding regions ----snp -epigenetic markers ---snp effect by environmental factors -----metalation patterns suspect--> leads
aims of current research of age estimation
validation of identified markers expanding prediction to -diff tissues -ethnic groups -younger individual -diseased populations
multiplex STR kits in use today take advantage of multiple fluorescent dyes
various dye colors are separated, and the peaks representing DNA fragments are identified and associated with the appropriate color. The DNA fragments are then sized by comparison to an internal sizing standard. the PCR product sizes for the questioned sample are correlated to an allelic ladder that has been sized in a similar fashion with internal standards.
