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(Str: dp. 16,100; 1. 466'0", b. 55'11", dr. 28'9", s. 11 k.; cpl. 70; a. 1 5", 1 3")
Roepat (Id. No. 2536), built in 1914 by William Hamilton & Co., Ltd., Glasgow, Scotland, for service as a Duteh animal transport ship, was seized by U.S. customs officials, taken over and commissioned as a Naval Overseas Transportation Service vessel 17 May 1918 at New York.
Following refit and refurnishing, Roepat loaded a ear~o of general Army supplies and sailed 2 June in convoy for Brest and St. Nazaire where she discharged her cargo. She returned to New York in convoy 30 July.
Loading Army supplies at Norfolk, she sailed in convoy from New York 17 August for Marseille where she arrived 6 September to unload her cargo. She returned to New York 18 October to undergo repairs and load Army cargo for Verdon where she arrived 18 November. She returned to Norfolk 22 December.
Loading a cargo for the Shipping Board Account at Baltimore, she bunkered at New Orleans and sailed from the latter port 5 February 1919 for Cette via Newport News. Diseharging her cargo of wheat and grain consigned to the Swiss Government 4 March, she returned to New York 6 May and underwent repairs.
Roepat sailed 24 May for Portland, Maine where she loaded a cargo for the Shipping Board and sailed on the 29th for Amsterdam where she arrived 28 June. On 30 June Roe pat was placed out of commission and returned to her owner, Nederland Stoomvaart Maats chappij.
Introduction to Short Tandem Repeat
I hope everyone is staying safe and healthy during these unfortunate times. My last post was in relation to being a new MLS grad and beginning my career as a molecular technologist at Northwestern University’s Transplant Lab. Time definitely flies by!
Today I’m going to provide a basic introduction on an assay I’ve recently been trained on called Short Tandem Repeat (STR). If you were to take a glance at your genome, it would be littered with many repeating sequences. While there are many different classifications of repeating sequences, STRs are a type of tandem repeating sequence where each repeat is approximately 2 to 7 nucleotides in length. 1,2,3 STR is well-known in forensic science to help identify a suspect at a crime scene when different sources of DNA are present. Yet, its applications are many – from cell line confirmation, paternal testing, and all the way to chimerism analysis! 4
Image 1. Electropherogram depicting two different alleles (11 and 17) within 1 locus (D6S1043). Allele 11 has 11 repeats and allele 17 has 17 repeats.
STRs are polymorphic, one useful characteristic among many, which make its utilization in identifying the source of DNA particularly advantageous. An STR allele is defined by the number of times the repeating sequence, defined above, repeats. (Image 1). 1 Individuals are either heterozygous or homozygous at each locus. As the number of STR loci being evaluated increases, the statistical power of discrimination increases and the likelihood of another individual having the same profile becomes increasingly unlikely and detecting small differences increases. 3,4 In our lab, we evaluate a total of 21 different loci!
Additionally, in our HLA lab we utilize STR to monitor chimerism status in patients who have undergone an allogeneic stem cell transplant. Before their transplant, patients are matched to a donor through their HLA system (different from STR). Once an HLA match is confirmed, we utilize the patient’s pre-transplant and the donor’s sample to generate STR informative alleles. Informative alleles are alleles that are present only in the recipient and not the donor. These alleles are important because stem cell transplants replace the recipient’s marrow and the detection of recipient DNA in post-transplant samples is crucial to identifying rejection or relapse of their disease. Additionally, loci that contain informative alleles are defined as informative loci, these are the loci then used to identify the percentage chimerism (Image 2).
Image 2. Donor and recipient (pre-transplant) are represented in the first two electropherograms. The green “D1R” tags represent shared alleles, the blue “D1” tags represent donor specific alleles, and the brown “R” tags represent recipient specific alleles. In the first locus, AMEL, there are no informative alleles and therefore it is not an informative locus. In the next locus, D3S1358, there is one shared allele, 15, one donor allele, 17, and one recipient informative allele, 18. Informative loci have recipient informative alleles and can detect the presence of recipient DNA in a sample – in this case all of the following loci after AMEL are informative loci. CD3 (post-transplant) is represented on the third electropherogram. In this example, it is clear that the patient is having some sort of graft failure or reoccurrence of their disease because their own cells, instead of just the donor, are present.
When recipient cells begin to re-emerge, we can detect the relative allele peaks and assign them to the recipient or donor by referring to the informative alleles. Allele peaks are then utilized through equations in our software that measure the area under these defined peaks and then compute a donor percentage chimerism. Once that informative report is created, we can compare any proceeding post-transplant sample to determine the patient’s chimerism status (Image 2).
Interesting enough, we don’t simply isolate the DNA from the post-sample buffy like we would with other samples. Rather, we separate each post sample into a total of three sub-samples. The first is simply the patient’s peripheral blood – nothing fancy. The other two are isolated from the rest of the peripheral blood that was not used into two separate cell lineages – lymphocytes (CD3+) and myelocytes (CD33+). This process is extremely labor-intensive, being able to process a set of up to 8-12 patients at a time and each set taking up to 4-5 hours.
The process begins by aliquoting peripheral blood for DNA isolation (sample 1) and taking the remainder and layering it over a lymphocyte separation medium (LSM). Then harvesting the lymphocyte/white cell layer from the spun down LSM. We then go on to add CD3 and CD33 antibody selection cocktails and magnetic beads. Then, we do a series of washes with magnets and eventually end up with our purified CD3 and CD33 cell populations. Their purity is determined through flow cytometry, an important component to confirm that our leukocyte subsets aren’t contaminated with other leukocyte populations – as contamination would defeat the purpose of analyzing different lineages.
Finally, we take the isolated DNA from the three sub-samples and amplify it with specific primers and fluorescent tags through PCR. Then the samples are loaded onto a capillary electrophoresis instrument. This instrument will detect each fragment length, defined by the primers and the repeats within, and be able to identify these fragments through size, fluorescent tags, lasers, and detectors. The instrument will then generate data that we can take to our analyzing platform, which is ChimerMarker. Through this we can analyze the data and generate our clinical reports.
Though extremely time intensive, lineage-specific chimerism is critical in stem cell transplant because it is more informative and sensitive than total leukocyte analysis – being several magnitudes more sensitive than analyzing just peripheral blood alone. It permits early detection of small chimeric cell populations that otherwise may go undetected, as one subset in the peripheral blood may “mask” another subset that has increasing percent recipient cells. Diagnosing these small cell chimeric cell populations as early as possible is critical for therapeutic interventions and reductions in graft rejections. 2,5,6
Furthermore, not only is their detection important, but through our analysis we can calculate the percentage of donor cells and recipient cells. We oftentimes report out the donor percentage (%) chimerism. For example, a patient at 322 days post-transplant could have a donor chimerism of 96% in their peripheral blood, 100% in their CD33 lineage, and 73% in their CD3 lineage. Then, at day 364 post-transplant they may then be at 100% in their peripheral blood, 100% in their CD33 lineage, and 92 percent in their CD3 lineage. Two things to notice in this example is that the percentages are changing (increasing in donor chimerism in this case) and that the peripheral blood expressed 100% chimerism in the second sample at 364 days, but when we look specifically at the CD3 sub-population at 364 days there was still 8% of recipient cells present (Image 3 & 4).
Image 3. Samples at 322 days post-transplant. Peripheral blood reports 96% donor chimerism. CD3 and CD33, purified from peripheral blood, reports 73% and 100% donor chimerism, respectively. Image 4. Samples at 364 days post-transplant, same patient as in Image 3 above. Peripheral blood reports 100% donor chimerism. CD3 and CD33, purified from peripheral blood, reports 92% and 100% donor chimerism, respectively.
Some studies have focused not only on the trends in percentages changing, but also in their relative percentage constellation. For example, one study found that increased recipient CD3 cells had an increased predictive factor of graft rejection. It was also found that further sub-leukocyte populations increased this predictive power. 5 Even more, there have been some studies that have looked at chimerism and its usefulness in predicting graft versus host disease (GvHD). This disease is defined by donor leukocytes attacking the leukocytes and tissues of the recipient. Through these and other findings, the potential and applicability of chimerism monitoring is extremely crucial to patient care during their transplant progression. 2,5,6,7
While engraftment is a very dynamic process, varying from individuals and disease-types, engraftment monitoring is one way to monitor and ultimately influence therapeutic approaches. 2,5,6 I am proud to be able to contribute to the wonderful team here at Northwestern University and I strive to learn more about the process – both clinical and in the lab. In future articles, I hope to go into more detail about the process and other assays that we perform.
Thanks for reading! Until next time! Stay well and safe during these uncertain times!
- Life Technologies. 2014. DNA Fragment Analysis by Capillary Electrophoresis. Thermo Fisher Scientific. http://www.thermofisher.com/content/dam/LifeTech/global/Forms/PDF/fragment-analysis-chemistry-guide.pdf.
- Kristt, D., Stein, J., Yaniv, I., & Klein, T. (2007). Assessing quantitative chimerism longitudinally: technical considerations, clinical applications and routine feasibility. Bone Marrow Transplantation, 39(5), 255–268. doi: 10.1038/sj.bmt.1705576
- Clark, J.R., Scott, S.D., Jack, A.L., Lee, H., Mason, J., Carter, G.I., Pearce, L., Jackson, T., Clouston, H., Sproul, A., Keen, L., Molloy, K., Folarin, N., Whitby, L., Snowden, J.A., Reilly, J.T. and Barnett, D. (2015), Monitoring of chimerism following allogeneic haematopoietic stem cell transplantation (HSCT): Technical recommendations for the use of Short Tandem Repeat (STR) based techniques, on behalf of the United Kingdom National External Quality Assessment Service for Leucocyte Immunophenotyping Chimerism Working Group. Br J Haematol, 168: 26-37. doi:10.1111/bjh.13073
- Short Tandem Repeat Analysis in the Research Laboratory. (2012). Retrieved April 10, 2020, from https://www.promega.com/resources/pubhub/short-tandem-repeat-analysis-in-the-research-laboratory/
- Breuer, S., Preuner, S., Fritsch, G., Daxberger, H., Koenig, M., Poetschger, U., … Matthes-Martin, S. (2011). Early recipient chimerism testing in the T- and NK-cell lineages for risk assessment of graft rejection in pediatric patients undergoing allogeneic stem cell transplantation. Leukemia, 26(3), 509–519. doi: 10.1038/leu.2011.244
- Buckingham, L. (2012). Molecular diagnostics: fundamentals, methods, and clinical applications. Philadelphia: F.A. Davis Company.
- Rupa-Matysek, J., Lewandowski, K., Nowak, W., Sawiński, K., Gil, L., & Komarnicki, M. (2011). Correlation Between the Kinetics of CD3 Chimerism and the Incidence of Graft-Versus-Host Disease in Patients Undergoing Allogeneic Hematopoietic Stem Cell Transplantation. Transplantation Proceedings, 43(5), 1915–1923. doi: 10.1016/j.transproceed.2011.02.011
-Ben Dahlstrom is a recent graduate of the NorthShore University HealthSystem MLS program. He currently works as a molecular technologist for Northwestern University in their transplant lab, performing HLA typing on bone marrow and solid organ transplants. His interests include microbiology, molecular, immunology, and blood bank.
The first DNA typing technology introduced in the mid 1980s was RFLP. The RFLP method of DNA typing involved core units of sequences consisting of 30 to 100 nucleotides which are present in many repeats (VNTR). The RLFP method of DNA typing requires intact genomic DNA in large quantities (20 to 30 mg). However, the biological specimens received in a forensic science laboratory are usually environmentally assaulted and occasionally only small amounts of DNA can be obtained. Hence in many situations, the RFLP method could not be applied.
The DNA typing method presently in use is STR typing. In this method many loci composed of core units of nucleotides repeated up to a length of 80 to 400 base pairs can be co-amplified and the results can be obtained in the same day by automated DNA fragment analyses. This technology is more superior than the RFLP method because it requires minute amounts of DNA (0.5 to 1 ng) and degraded samples can also be tested.
DNA analysis has been instrumental in securing convictions in hundreds of violent crimes, from homicides to assaults. It has also helped to eliminate suspects and has led to the exoneration and release of previously convicted individuals. DNA can focus investigations, and will likely shorten trials and lead to guilty pleas. It could also deter some offenders from committing serious offences. The increased use of forensic DNA evidence will lead to long-term savings for the criminal justice system.
Through storing DNA data in computer data banks, DNA analysis can be used to solve crimes without suspects. Forensic scientists can compare DNA profiles of biological evidence samples with a data bank to assist the police in detecting suspects. A data bank would also enable unsolved earlier offences where DNA evidence had been found but not linked with the offender, to be cleared up if DNA samples taken from a suspect in connection with a later offence matched the evidence found at the scene of the earlier crime. A national DNA data bank would also help police identify serial offenders both within and across the country.
Forensic DNA analysis is conducted throughout the world. Hence it is imperative on the part of the developing nations including Malaysia to develop and compile a national DNA database consisting of 𠇌rime scene DNA profile index”, 𠇌onvicted offender DNA profile index”, and an index containing DNA profiles of unidentified bodies and body parts. This effort in turn will warrant appropriate amendments in criminal laws to help law enforcement agencies identify persons alleged to have committed serious and violent offences and empowering collection of samples for DNA profiling database. To date, there is already published data for 9 STRs for three ethnic population groups of Malaysia (Malay, Chinese and Indians) (21, 22) and efforts are currently underway to type subpopulations of Malays and to start the newly validated, 15 STR profiling kit in various populations in Malaysia. Extensive database and DNA profiling of criminals and indexing them will help to speed up crime detection.
How DNA Evidence Works
From the crime scene, a piece of DNA evidence travels to a forensic laboratory. These labs vary quite a bit, both in terms of how they are structured and what kind of analyses they offer. Public laboratories are often associated with a law enforcement entity or the district attorney's office, while others are independent government entities. Private forensic laboratories, some dedicated just to DNA analysis, also exist.
Many labs have the ability to conduct testing on nuclear DNA, which is the copy of DNA that exists in the nucleus of every cell. But only a few labs offer more specialized techniques, such as Y-chromosome or mitochondrial DNA analysis. Let's look at some of these techniques in greater detail.
Restriction fragment length polymorphism (RFLP) analysis was one of the first forensic methods used to analyze DNA. It analyzes the length of strands of DNA that include repeating base pairs. These repetitions are known as variable number tandem repeats (VNTRs) because they can repeat themselves anywhere from one to 30 times.
RFLP analysis requires investigators to dissolve DNA in an enzyme that breaks the strand at specific points. The number of repeats affects the length of each resulting strand of DNA. Investigators compare samples by comparing the lengths of the strands. RFLP analysis requires a fairly large sample of DNA that hasn't been contaminated with dirt.
Many laboratories are replacing RFLP analysis with short tandem repeat (STR) analysis. This method offers several advantages, but one of the biggest is that it can start with a much smaller sample of DNA. Scientists amplify this small sample through a process known as polymerase chain reaction, or PCR. PCR makes copies of the DNA much like DNA copies itself in a cell, producing almost any desired amount of the genetic material.
Once the DNA in question has been amplified, STR analysis examines how often base pairs repeat in specific loci, or locations, on a DNA strand. These can be dinucleotide, trinucleotide, tetranucleotide or pentanucleotide repeats -- that is, repetitions of two, three, four or five base pairs. Investigators often look for tetranucleotide or pentanucleotide repeats in samples that have been through PCR amplification because these are the most likely to be accurate.
The Federal Bureau of Investigation (FBI) has chosen 20 specific STR loci to serve as the standard for DNA analysis. They expanded that number from 13 to 20 in January 2017.
STR typing: method and applications
This month’s foray by The Primer into molecular diagnostics techniques will cover a method known as short tandem repeat (STR) typing. STR typing is a method most commonly applied for molecular forensics work, but it is increasingly used as well in the molecular pathology lab as a method to use (or fall back on, as needed) for tracking and/or confirming “identity” of tissue samples. While the former application gets more screen time in TV and movies, it’s in the latter context that more readers will likely encounter the method. However as we’ll see, the actual method is identical in both uses.
Understanding short tandem repeats
We start by considering what an STR actually is. Across the human genome, there are large numbers of places (“loci”) where non-coding DNA sequences consist of a short (usually 3 or 4) nucleotide element, such as “CGG” or “ACAG,” which occurs as a set of concatameric repeats. For our examples, this would be “CGGCGGCGG….CGG” and “ACAGACAGACAG….ACAG,” respectively. When DNA replication occurs through these types of elements, a particular type of error can occur: if the nascent (growing) strand becomes detached from the template momentarily, when it re-anneals it can effectively do so with a “slippage” by some unit number of repeats, allowing almost all of the base pairing to re-establish. That is, after denaturation and reannealing of nascent strand to template, the polymerase may be “ahead of ”or “behind” where it was when it left. As replication starts again, the nascent strand then has either “skipped over” some of the template repeats or read them as template twice. The first results in a daughter DNA strand with a loss of a unit number of the STR repeats, while the second yields a daughter strand with an increased number of STR repeats.
How often does this polymerase “slippage” occur in an STR region? The frequency can vary with a number of factors, but the general answer is: rare, but frequently enough such that a population will display a range of STR repeat numbers at a given locus.
While STR loci occur scattered throughout the human genome, a small number are particularly well understood. These are ones which occur at a region flanked by highly conserved unique sequences, such that the unique sequences are not too close together, and not too far apart, for effective PCR amplification based on primers against the unique regions (roughly, 50 to 500 base pairs). When an STR element like this exists, it is possible to design PCR primers against the flanking conserved regions and know that (thanks to conservation) the primer set will amplify a product from any intact human DNA sample. Actually, when the STR loci in question are on an autosome as most are, then a human DNA sample will have two loci copies (one from maternally derived DNA, one from paternally derived), so two PCR products will be amplified. The important detail here is that while we know a PCR product (or two products) will form, we don’t know a priori how long the products will be. That length will depend on the number of STR repeats between the PCR primer sites. The most useful STR loci are ones where population studies have shown a wide diversity of number of repeats encountered—say, 5 to 30. This range of 25 “repeat numbers” for a trinucleotide repeat element would lead to a 75-base pair range (i.e., 25 x 3) of possible amplicon sizes, in steps of three bases per repeat.
Known by loci names such as “D1S80” or sometimes for nearby genes “TPOX,” a particular handful of STR loci meet these utility criteria and are well studied, with published primer sets for their amplification and known population statistics for individual repeat numbers at each locus—that is, for a locus, in a given ethnic population, some repeat numbers are commonly found, and others are less commonly found.
Finding a DNA fingerprint
With this in mind, imagine we take a primer set for an autosomal STR locus, and perform a simple endpoint PCR on a human sample. At the end of the PCR, we analyze the PCR products for size. Since we’re looking for 3-nt or 4-nt steps, gel electrophoresis won’t give us good enough resolution to distinguish products differing by one or two repeat numbers, so we employ capillary electrophoresis sequencers to read the product sizes exactly to the nucleotide. Our sample may show a single size product (indicating both loci copies had the same repeat number), or it may show two products, differing in repeat numbers (Figure 1). For the loci examined, we now know the repeat numbers associated with that DNA sample.
Now imagine we take a second DNA sample, and repeat the experiment. If we do so, and we get a different result than on the first sample, we have the inescapable conclusion that the two DNA samples come from different individuals. If instead we get the same results as the first sample, we cannot, however, say that the samples are from the same individual. That’s because it’s possible (to some statistical level, depending on the population frequency of the result obtained for this locus) that another person had the same repeat numbers as these loci.
The technique gains power when we test multiple STR loci in each specimen. The likelihood of two samples exactly matching decreases rapidly as we examine more loci. One commonly used commercial STR test examines 16 loci (15 autosomal, and one special case sex chromosomal discussed further below) with claimed specificity rates (that is, likelihood of two individuals matching all loci) of 1 in 1.8×10 17 individuals or more—that is, many times the total human population of the planet! Commonly referred to as a “DNA fingerprint,” with probabilities of that scale, it is little wonder DNA evidence is increasingly applied in forensic and criminal uses—both in popular entertainments and in real-life forensics.
In addition to determining (or disproving) identity between two samples, the technique can also be used to determine relatedness. Half of any individual’s STR loci repeat numbers should match values from the maternal source, and the other half from the paternal source. Siblings should generally match each other on one-fourth of loci, and so on, through simple Mendelian genetic relationships. Detailed statistical analysis (including population frequency of the particular STR types observed) can be employed to refine data of this sort and prove or disprove levels of relatedness between samples, to a known statistical probability. Note that since de novo STR slippage events and/ or experimental errors can occur, a single mismatch to expected values does not definitively disprove relatedness a match across the remaining loci may still be enough to have a high certainty of relatedness.
Now that we understand how this methodology is applied to the popular applications, what about the more mundane? The power and simplicity of the STR “fingerprinting” method, combined with its ready availability in pre-made optimized kit formats easily run on lab equipment already generally present in the molecular pathology laboratory, makes it a useful tool for tracking and/or confirming relatedness in samples (or pieces of samples). Consider a case such as a potential tumor biopsy, where multiple small tissue pieces may be embedded in a single FFPE block. Routine immunohistochemistry analysis is done, and the result shows most of the tissue pieces are non-cancerous, while a single small piece is. In cases such as this, a concern crossing the pathologist’s mind may be whether all of the tissue pieces are in fact from the same sample—or has a “floater” from another case somehow gotten into the block? While carefully guarded against, such cases are not impossible and can have serious consequences for the patient. The STR typing method can be a very useful tool in a case such as this, where a microscopic section of the tissue piece in question can serve as a template for one DNA fingerprint, with a reference sample from the patient providing another. A match confirms the “relatedness” (or not) of the tissue piece and the patient, assuring correctly assigned diagnosis.
A particular STR locus on the sex chromosomes was alluded to above. Occurring within the amelogenin gene, this isn’t strictly a classical STR where the size of a repeat element can vary rather, it turns out that the version of the amelogenin gene carried on the X chromosome (AMELX twice in females and once in males) is not precisely length-identical with the same region of the amelogenin gene carried on the Y chromosome (AMELY once in males). An intron in AMELY contains a 6-base insertion relative to the same intron in AMELX. (Note that since the insertion is in an intron, the coding gene portions are identical.) When amplified by primers flanking this region, AMELY-derived products are thus 6 bp longer than AMELX-derived. The most commonly used primer set for this locus thus yields a single 106 bp product (two loci copies, same size) for DNA samples from females, and a 106/112 bp doublet product (one from each locus) from males. This single locus test thus effectively rules in (or out) about half the population as possible sources for any given DNA sample, and is routinely included in STR typing panels. Given its size, this locus is also just barely differentiable on an agarose gel with good technique, making it a good classroom demonstration of the overall approach, without need for access to a capillary sequencer instrument.
What about mixed samples? The method described above has assumed each sample tested is from a single source, and works best in this “perfect” situation. However, in real-life cases with small proportionate amounts of other DNA present, the method still works. The primary template will yield the majority of product, with small amounts of product arising from the contaminating template. Capillary sequencers show the actual peak area for each product detected, so the primary sample will show a major set of peaks with small side peaks attributable to contaminant.
Return to Figure 1: a sketch of hypothetical STR results for four STR markers “A,” “B,” “C,” and “D.” Samples 1, 2, and 3 represent three different specimens tested for these markers, as the raw capillary electrophoresis results. Each sample contains two fixed size standards, “R1” and “R2,” which are used to ensure alignment of the results between samples. Each electropherogram itself shows signal strength vs. product size, and is divided into regions “A,” “B,” “C,” and “D.” Each region represents the expected range of possible amplicon sizes from the STR marker of the same name. Considering Sample 1, it can be seen that the source is heterozygous for STR sizes at markers A, C, and D, (two distinct products formed at specific sizes) but is homozygous for both B marker alleles. (There are two products but of identical size, yielding a single peak.) Sample 2 would have very low genetic relatedness to Sample 1 note that in this sample, STR markers B, C, and D are heterozygous while A is homozygous, and that in general few of any of the allele sizes line up between Samples 1 and 2. By contrast, Sample 3 is exactly the same as Sample 1, suggesting identity (or at least, a high degree of relatedness). A real STR result would look similar to this but with more markers, allowing for greater statistical power in detecting unrelatedness.
John Brunstein, PhD, a member of the MLO Editorial Advisory Board, is President and CSO of British Columbia-based PathoID, Inc.
Roepat Str - History
Most of our DNA is identical to DNA of others. However, there are inherited regions of our DNA that can vary from person to person. Variations in DNA sequence between individuals are termed "polymorphisms". As we will discover in this activity, sequences with the highest degree of polymorphism are very useful for DNA analysis in forensics cases and paternity testing. This activity is based on analyzing the inheritance of a class of DNA polymorphisms known as "Short Tandem Repeats", or simply STRs.
STRs are short sequences of DNA, normally of length 2-5 base pairs, that are repeated numerous times in a head-tail manner, i.e. the 16 bp sequence of "gatagatagatagata" would represent 4 head-tail copies of the tetramer "gata". The polymorphisms in STRs are due to the different number of copies of the repeat element that can occur in a population of individuals.
D7S280 is one of the 13 core CODIS STR genetic loci. This DNA is found on human chromosome 7. The DNA sequence of a representative allele of this locus is shown below. This sequence comes from GenBank, a public DNA database. The tetrameric repeat sequence of D7S280 is "gata". Different alleles of this locus have from 6 to 15 tandem repeats of the "gata" sequence. How many tetrameric repeats are present in the DNA sequence shown below? Notice that one of the tetrameric sequences is "gaca", rather than "gata".
We have made and will continue to make great efforts to ensure the accuracy and completeness of the data included in this STR database. Information will be added from time-to-time to keep this site as up-to-date as possible. The National Institute of Standards and Technology (NIST) is in no way responsible for information provided through this site, including hyperlinks to commercial sources of materials. Certain commercial vendors are identified in this web site to benefit the DNA typing community. In no case does such identification imply a recommendation or endorsement by NIST nor does it imply that the material, instrument or equipment identified is necessarily the best available for human identity testing.
All users agree that all access and use of this web site and on any site linked to this one and the content thereof is at their own risk. Neither NIST nor the webmaster for the STR DNA Internet Database assume responsibility or liability for the content of pages outside of this web site.
The Geriatric Patient
Steven J. Schwartz MD , Frederick E. Sieber MD , in Anesthesia and Uncommon Diseases (Sixth Edition) , 2012
Diagnosis and Differential
The DNA-repeat expansion forms the basis of a diagnostic blood test for the disease gene. Patients having 38 or more CAG repeats in the Huntington's disease gene have inherited the disease mutation and will eventually develop symptoms if they live to an advanced age. Each of their children has a 50% risk of also inheriting the abnormal gene a larger number of repeats is associated with an earlier age at onset. Huntington's can also be diagnosed by caudate atrophy on magnetic resonance imaging (MRI) in the context of an appropriate clinical history.
Differential diagnosis of Huntington's disease includes other choreas, hepatocerebral degeneration, schizophrenia with tardive dyskinesia, Parkinson's disease, Alzheimer's disease, and other primary dementias and drug reactions.
Performing STR Analysis
This method differs from RFLP since in STR analysis DNA is not cut with restriction enzymes. Probes are attached to preferred regions on the DNA, and a PCR is employed to discover the lengths of the short tandem repeats.
The whole process for STR typing comprisesof collection of sample, extraction of DNA, quantisation of DNA, amplification of multiple STR loci by PCR, separation and sizing of STR allele, STR typing followed by profile interpretation, and possibly a report of the statistical significance of a match. Current forensic systems apply 10 (e.g. United Kingdom) or 13 (e.g. United States) STR loci. Kits with PCR primers for the standard STR loci are available commercially.
Numerous PCR reactions are performedconcurrently in a single tube at different STR loci, which give several products (two for each locus). The required components are as follows:
§ A DNA sample, e.g. blood or buccal cells from a suspect or a tissue, hair, nail from the scene of a crime
§ Two oligonucleotide PCR primers: one unlabelled reverse primer and one primer labelled at the 5 ′ -end with 32 P
§ A DNA polymerase (thermos table)
§ Four deoxynucleoside triphosphates which are as follows: dATP, dGTP, dCTP, dTTP.
The labelled PCR products separate according to size when run on a polyacrylamide gel. DNA ladder is obtained and works as characteristic of an individual (Fig.7) .
Repeat specific rows or columns on every printed page
If a worksheet spans more than one printed page, you can label data by adding row and column headings that will appear on each print page. These labels are also known as print titles.
Follow these steps to add Print Titles to a worksheet:
On the worksheet that you want to print, in the Page Layout tab, click Print Titles , in the Page Setup group).
Note: The Print Titles command will appear dimmed if you are in cell editing mode, if a chart is selected on the same worksheet, or if you don’t have a printer installed. For more information about installing a printer, see finding and installing printer drivers for Windows Vista. Please note that Microsoft has discontinued support for Windows XP check your printer manufacturer's Web site for continued driver support.
On the Sheet tab, under Print titles, do one—or both—of the following:
In the Rows to repeat at top box, enter the reference of the rows that contain the column labels.
In the Columns to repeat at left box, enter the reference of the columns that contain the row labels.
For example, if you want to print column labels at the top of every printed page, you could type $1:$1 in the Rows to repeat at top box.
Tip: You can also click the Collapse Popup Window buttons at the right end of the Rows to repeat at top and Columns to repeat at left boxes, and then select the title rows or columns that you want to repeat in the worksheet. After you finish selecting the title rows or columns, click the Collapse Dialog button again to return to the dialog box.
Note: If you have more than one worksheet selected, the Rows to repeat at top and Columns to repeat at left boxes are not available in the Page Setup dialog box. To cancel a selection of multiple worksheets, click any unselected worksheet. If no unselected sheet is visible, right-click the tab of a selected sheet, and then click Ungroup Sheets on the shortcut menu.