MDX-DNA POLYMORHISM AND HUMAN IDENTIFICATION

17. STR locus information

CD4-12p, AAAAG CSF1PO-5q, TAGA D3S1358-3p, TCTA D5S818-5q, AGAT D7S829-7q, GATA D8S1179-8q, TCTA D13S317-13q, TATC D16S539-16q, GATA D18S51-18q, GAAA D21S11-21q, TCTG F13A01-coagulation factor IX-6p, GAAA F13B-factor XIIIb-1q, ttta FESFPS-c-fes/fps protooncogene-15q, ATTT HPRTB-hypoxanthine phosphoribosyl-transferase-Xq, TCTA LPL-lipoprotein lipase-8p-TTTA TH01-tyrosine hydroxylase-11p-tcat TPOX-thyroid peroxidase-2p, TGAA vWA-von Willebrand factor-12p-TCTA Penta D-21q, AAAGA Penta E-15q, AAAGA

19. Commercial STR kits

CODIS=FBI, combined DNA indexing system, 13 core loci: TPOX, D3S1358, FGA, D5S818, CSF1PO, D7S820, D8S1179, TH01, vWA, D13S317, D16S539, D18S51, D21S11, and the amelogeni locus on the X and Y loci NIST=natl institute of standards and technology, supplies standard material that certifies values for 22 STR loci, including CODIS and marjers used by European forensic laboratories Profiler Plus (Life technology/ABI) and PowerPlex (Promega) primer mixes include the CODIS loci

11. STR typing by PCR

HLA DQ alpha system or DQA1 system (Cetus corporation, 1986): could distinguish28 DQA1 types Polymarker (PM system: could type 5 additional genetic markers; PM system is a set of primers complimentary sequences flanking short tandem repeats (STRs) or microsatellite STRs are similar to VNTRs (minisatellites) but have repeat units of 1 to 7 bp because of the increased power of discrimination and ease of use of STR, the HLA DQA forensic amplification and typing kit was discontinued in 2002 most STRs used have 4-5 bp occasionally, STRs contain repeat units with altered sequences, or microvariants, repeat units missing one or more bases of the repeat smaller STRs are efficiently amplified by PCR long, intact DNA fragments are not required to detect the STR products, therefore, degraded or otherwise less-than-optimal specimens are potentially informative

3. Types of useful polymorphisms and lab methods

RFLP-one or more nt changes that affect the size of RE enzyme products-SB VNTR-repeats of 10-50 base sequences in tandem-SB, PCR STR-repeats of 1-10 base sequences in tandem-PCR SNP-alterations of a single nt-Sequencing, other...

30. second phase of transplantation phase: engraftment phase

after conditioning and infusion with the donor cells, the patient enters the engraftment phase, in which the donor cells reconstitute the recipient's bm once a successful engraftment of donor cells has been established, the recipient is a genetic chimera (the recipient has body and blood cells of separating genetic origin)

18. Gender identification

amelogenin locus=a useful marker, not an STR, located on intron 2 of chromosome X and Y amelogenin is required for embryonic and tooth maturation Y-alle is 6 bp larger than the X-alle male-XY- 2 bands or peaks, Y-allele =218 bp female-XX-1 band or peak, X-allele = 212 bp some commercial STR kit include amelogenin polymorphsm in addition to STR primer sets

36. Specimens for engraftment analysis

bm specimens can be taken at the time of bm biopsy following the transplant blood specimens are taken in intervening periods 3-5 mL of bm or 5mL of blood is more than sufficient for analysis specimens collected soon after the transplant may be hypocellular, so larger volume 5-7 mL bm or 10-20 mL blood may be required

14. Mini-STRs

these STR are amplified with PCR primers located closer to the tandem repeat than in standard STR the small amplicons are more efficiently produced from such challenging materials such as fixed tissues and degraded specimens

4. RFLP typing

used for gene mapping, human ID, parental testing observed as differences in the sizes and number of fragments generated by RE digestion of DNA fragment sizes vary because of the changes in nt sequence in or between recognition sites of RE nt change may also destroy, change, or create RE sites,altering the numbers of fragments

35. Pre-transplant and engraftment analyses

(1) early method: amplification of VNTRs and resolution of amplified fragments on PAGE with silver-stain detection before transplant: the screen for informative loci ws based on band patterns of the PCR products after transplant: analysis of the band patterns from the blood and bm of the recipient revealed one of 3 different states: full chimerism (FC, in which the donor alleles were detected in the recipient); mixed chimerism (MC, in which a donor and recipient alleles was present; or graft failure (GF, in which only recipient alleles were detectable) (2) current method: amplification of STRs, resolution by CE and fluorescent detection; ease of use, accurate quantification of the % of donor/recipient cells, and high sensitivity with minimal sample requirements (3) donor and recipient DNA for allele screening prior to transplant can be isolated from blood or buccal cells; 1 ng of DNA is sufficient for the screening of multiple loci; however, 10 ng is a more practical lower limit (4) multiple loci can be screened simultaneously using multiplex PCR (5) primer sets that specifically amplify Y-STR may also be useful for sex-mismatched donor recipient pairs (6) the five tetramethylrhodamine (TMR)-labeled loci from the PowerPlex system is used for the identity testing are vWA, TH01, AMEL, TPOX, and CSFIPO (7) a total of 9 loci are amplified using this set of multiplex primers (8) all loci may not amplify with equal efficiency in a multiplex reaction, e.g. the amelogenin locus did not amplify as well as the other 4 loci in the multiplex; this can be seen from the lower peak heights in the amelogenn products compared with the other products of the other primers (9) investigating peak sizes and areas is done with fragment analysis and sometimes required adjustment of the instrument or capillary polymer (10) automatic detection will generate an electropherogram; informative and non-informative loci will appear as nonmatching or matching donor recipient peaks, respectively, and many combinations of donor-recipient peaks are possible; optimal loci for analysis should be clean peaks without stutter, esp. stutter peaks that comigrate with informative peaks, non-specific amplified peaks (misprimes), or other technical artifacts (11) the chosen locus should have at least one recipient informative allele to assure direct detection of minimal amounts of residual recipient cells; if the recipient is male and the donor is female, the amelogenin locus supplies a recipient-informative locus; good separation (by 2 repeat units) of the recipient and the donor alleles is desirable for ease of discrimination in the post-transplant testing; the choice of informative alleles are more limited in related donor-recipient pairs, as they are likely to share alleles; unrelated donor-recipient pairs will yield more options (12) after the transplant, the recipient is tested on a schedule determined by the clinician or according to concensus recommendations; with non-myeloblative protocols, testing is recommended at 1, 2, 6, and 12 months ; because engraftment may predict GVHD or graft failure after nonmyeloblative treatments, even more frequent blood testing may be necessary, such as at 1, 2, and 3 months after the transplant

10. RFLP SB technique

100 ng to 1 ug of relatively high quality DNA, 1-20 kbp, large, fragile, 0.7% gels were required to achieve adequate band resolution, and the 32P-based probe system could take 5-7 days to yield clear results the probability of 2 people having the same set of RFLP or profile becomes lower and lower as more loci were analyzed

44. Single nucleotide polymorphisms (SNP)

HGP: human nt sequence differs every 1000-1500 bases from one individual to another majority of these sequence differences are variations of single nt or SNPs traditional definition of polymorphisms: requires that the genetic variations be present at a frequency of at least 1% of the population International SNP Map Working Group: 2 haploid genomes differ at 1 nt per 1331 bp; predicts 11 million sites in a genome of 3 billion bp that vary at least 1% of the world's population (i.e., each individual has 11 million SNPs) So far, ~ 5 million SNPs have been identified; 99% of these have no biologival efefct; over 60,000 are within genes and some are associated with diseases

16. STR Nomenclature

ISFG (international society for forensic genetics) recommended nomenclature for STR loci in 1997 STRs within genes are designated according to the gene names e.g. TH01 = in intron1 of human tyrosine hydroxylase on chromosome 11 TPOX = in intron 10 of human thyroid peroxidase gene on chromosome 2 these STRs do not have any phenotypic effect with respect to these genes non-gene associated STRs are designated by the D#S# system D-DNA # after D is chromosome number S= unique segment # after S is a registered number of S in GDB (international genome database) e.g. D5S818: DNA, chromosome 5, unique segment #818

45. Types of SNPs

Type I-coding region-nonconservative Type II-coding region-conservative Type III-coding-silent Type IV-non-coding 5'UTR Type V-noncoding 3'UTR Type 6-noncoding, other

25. Bone marrow engraftment (BME)/transplantation (BMT)

a method used to treat malignant and nonmalignant blood disorders and some solid tumors

13. RFLP probes

at least 3-7 RFLP probes were required to determine genetic identity available probes include: G3, MS1, MS8, MS31, and MS43, which were subclones of Jeffrey's multilocus probes 33.6 and 33.15 and pYNH24m, MS205, and MS621 single locus probes MS1, MS31, MS43, G3, and YNH24 were used by Cellmark in the OJ Simpson trial in 1996

27. Myeloablative transplant strategies

high doses of therapy completely remove the recipient bm, particularly the stem cells that give rise to all other cells in the marrow (conditioning); the allogenic or autologous cells are then expected to re-establish a new bm in the recipient (engraftment) more toxic appraoch

41. Technical issues

some amplification artifacts occur during PCR some polymerases add an additional nontemplate A residues to the 3'-end of PCR prodcut if this 3'nt addition does not include all the ampligied products, a mixed sets of amplicons will result in extra bands or peaks located very close together Stutter is another anomaly of PCR amplification, in which the polymerase may miss a repeat during the replication process, resulting in 2 or more different species in the amplified product. These also appear as extra bands or peaks; generally the larger the repeat unit length, the less stutter observed These or other aberrant band patterns confuse the analysis software and can result in the miscalling of alleles

38. Cell lineages

some cell fractions such as granulocytes engraft before tohers; e.g., isolated granulocytes may show full chimerism while the T-cell fractions still showed mixed chimerism; this is a case of split chimerism because cell lineages engraft with different kinetics, testing of blood and bm may yield different levels of chimerism; bm will contain more myeloid cells, and blood will contain more lymphoid cells in mixed chimerism, cell separation techniques may be used to determine which lineage are mixed and which are fully donor nonmyeloablative conditioning of the transplant recipients requires monitoring of both myeloid and lymphoic cell engraftment

37. Quantification of engraftment

% recipient and donor cells at post-transplant is oerformed using the informative locus or loci selected dring the pre-transplant informative analysis the raw data for these calculations are the areas under the peaks generated by PR products after amplification; the emission from the fluorescent dyes atatched to the primers and thus to the ends of PCR products is collected as each product migrates past the detector; the fluorescent signal is converted into fluorescent units by the software; the software displays the PCR prodcuts as peaks of fluorescent units (y-axis) versus migration speed (x-axis) the amount of fluorescence in each product or peak is represented as the area under the peaks for homozygous or heterozygous donor and recipient peaks with no shared alleles, % of recipient cells = R/(R+D), R=area under recipient-specific peak(s), D=area under the donor-specific peak(s) shared alleles, where one allele is the same for donor and recipient can be dropped from the calculation, and % of recipient cells is calculated as: R(unshared)/(R(unshared) + D(unshared) Chimerism/engraftment rsults are reported as percent recipient cells and/or percent donor cells in the bm, blood, or cell fraction

34. Engraftment/chimerism DNA testing

(1) pre-transplant analysis: several polymorphic loci in the donor and recipient cells must be screened to find at least one informative locus, i.e. one locus in which donor alleles differ from the recipient alleles non-informative loci are those in which the donor and recipient have the same alleles in donor-informative loci, donor and recipient share one allele for which the donor is heterozygous and the donor has a unique allele in recipient-informative loci, the unique allele is in the recipient (2) engraftment analysis is performed at specified intervals after the transplant; the recipient blood and bm are tested to determine the presence of donor cells using the donor-informative and/or recipient-informative loci

46. Human haplotype mapping (HapMap) project

2 people are 99.5% identical at the DNA sequence level the other o.5% difference is important, may be basis of differences in diseases susceptibility and other variations among normal human traits linked markers: genes, RFLPS, VNTRs, STRs have been mapped previously blocked of closely linked SNPs on the same chromosome tend to be inherited together; recombination rarely takes places within these sequences (linkage disequilibrium) all the SNPs on that block comprises a haplotype SNP haplotypes tend to be 20,000-60,000 bp of DNA sequences containing up to 60 SNPs; this also means that up to 60,000 bp of sequences can be identified through detection of 4 or 5 informative SNPs or tag SNPs SNP haplotypes offer great potential for mapping of disaese genes the disease allele and he SNPs closest to it (the haplotype) tend to be inherited as a group'\; this haplotype should always be present in patients with the disease Human haplotype mapping project (HapMap, 2002-2005) goal is to map the common patterns of SNPs in the form of a haplotype map or HapMap SNPs are detected by Bead Array, Invader, Multiplex inversion probe, fluorescent polarization-templete directed dye terminator incorporation, and homogenous mass extend (mass array)

47. Mitochondrial DNA polymorphism

a circular genome of 16,569 bp the 2 strands of circular mtDNA have an asymmetric distribution of gs and Cs generating a G-rich heavy (H) anda C-rich light (L) chain each strand transcribed from a control region starting at one predominant promoter, PL on the L strand and PH on the H strand, located in the sequence of the mitochondrial circle called the displacement (D-loop) the D-loop forms a tripke-stranded region with a short piece of H-strand DNA, the 7S DNA, synthesized from the H-strand Bi-directional transcription starts from PL on th L-strand and PH1 and PH2 on the H-strand RNA synthesis proceeds aroung the circle in boh directions a bidirectional attenuator sequence limits L-strand synthesis and doing so maintains a high ratio of rRNA to mRNA transcripts from the H-strand mature mtRNA, 1 to 17, are generated by cleavage of polycistronic (multiple gene)transcript at the location of tRNA genes mtDNA genes: 22 tRNA genes, 2 rRNA genes, and 12 genes coding for components of the OX/PHOS system mutations in these genes are responsible for neuropathies and myopathies mtDNA has 2 noncoding regions that vary in DNA sequence and are called hypervariable region I and hypervariable region II or HVI and HVII the reference mtDNA hypervariable region is the sequence published initially by Anderson, called the Cambridge reference sequence, the Oxford sequence, or the Anderson reference polymorphisms are denoted as variations from the reference sequence mtDNA follows maternal clonal inheritance pattern; i.e., mtDNA are inherited maternally these characteristics make possible collection of reference material for forensic analysis, even in cases in which generations are skipped the quality of mtDNA match between 2 mtDNA sources by counting the number of times the mtDNA profile ocurs in data collections of unrelated individuals, so the estimate of the uniqueness of a particular mtDNA type depends on the size of the reference database

22. Y-chromosome mutation

a low mutation rate (1.72-4.27 mutations/1000 alleles assuming the Y chromosome mutation rate is 1/every 500 generations/locus, for 25 loci, 1 locus should have a mutation every 20 generations (500 generations/25 markers=20 generations) lineage testing over several generations is possible due to this low mutation rate it is also useful for "missing person" cases in which reference samples can be obtained from paternally related males

28. Non-myeloblative transplant

also called mini-transplants pre-transplant therapy will not completely remove the recipient bm; the donor bm is expected to eradicate the remaining recipient cells through recognition of residual recipient cells as foreign to the bm the procedure also imparts a graft-versus-leukemia or graft-versus-tumor (GVT) effect, closely related to graft-versus-host diseases (GVHD) T-cells fraction of the donor marrow is important for engrafment and for GVT effect; efforts to avoid GVHD by removing the T-cell fractions before infusion of donor cells have resulted in increased incidence of graft failure and relapse

20. Analysis of STR test results

analysis of polymorphism at multiple loci resulst in high levels of discrimination 8 loci: ~1/10E9 16 loci: ~1/10E18 discovery of the same set of alleles from different sources or shared alleles between allegedly related individuals is strong evidence of identity, paternity, or relatedness results must be expressed in terms of probability of chance matches DNA testing results in peaks or band patterns that must be converted to genotype (allele identification) for comparing results between laboratories STR locus genotype is defined by the number of repeats in the alleles if the locus genotype represents homologous chromosomes from an. individual, the locus would be heterozygous, with 7 repeats on one chromosome and 8 repeats on the other, this locus would designate as 7/8 or 7,8 a homozygous locus, where both homologous chromosomes carry the same allele, is designated by the single number of repeats of that allele; e.g. 7/7 or 7,7 some reorts use a single number, such as 6 or 7, to designate a homozygous locus microvariant alleles containing partial repeat ubits are indicated by the number of complete repeats followed by a decimal point and then the number of bases in the partial repeat e.g. the 9.3 allele of the TH01 locus has 9 full 4-bp repeat units and 1 repeat unit with 3 bp microvariants are detected as bands or peaks very close to the full-length allele the genotype or profilr of a specimen is the collection of alleles in all the locus genotypes tested a matching genotype is not necessarily an absolute determination of an individual genetic concordance is a terms used to express the situation where all locus genotypes (alleles) from 2 sources are the same concordance is interpreted as inclusion of a single individual as the donor of both genotypes 2 samples are considered different if at least one locus genotype differs (exclusion)

26. Types of BME/BMT

autologous transplant (from self): in which cells from the patient's own bone marrow are removed and stored; patient then receives high doses of chemotherapy and/or radiotherapy; the patient's marrow may also be purged of cancer cells before being returned to the patient allogenic transplant(between 2 individuals): the donor supplies healthy cells to the recipient patient. Donor cells are supplied as bone marrow, peripheral blood stem cells (also called hemopoietic stem cells) or umbilical cord blood. To ensure successful establishment of the transplant donor cells, the immune compatibility of the donor and the recipient is tested before transplant by HLA typing

21. Y-STRs

autosomal STRs: each locus is defined by 2 alleles, one from each parent Y-STRs: are represented only once per genome and only in males a set of Y-STRs alleles comprises a haplotype, or series of linked alleles always inherited together (b/c the Y chrom cannot exchange information (recombine) with the X chrom or the other Y chrom marker alleles on the Y chrom are inherited from generation to generation in a single block therefore, the frequency of the entire Y-STR profiles (haplotypes) in a given population can be determined by empirical studies e.g. if a combination of alleles (haplotype) was observed only 2 times in a test of 200 unrelated males, that haplotype is expected to occur with a frequency of 2/200=1/100males test in the future the discrimination power f Y-haplotype testing will depend on the number of subjects tested and will always be less definitive than that of autosomal STR polymorphism on Y chrom, however, have unique characteristics that have been exploited in forensic, lineage, population studies, and kinship testing every male member of a family (brothers, cousins, uncles, and grandfathers) will have the same Y-chromosome haplotype Y-chromosome inheritance can be applied to lineage, population and human migration studies Y-STR/paternal lineage test can determine whether 2 or more males have a common paternal ancestor a paternal lineage tests serves as supportive evidence for adoptees, and their biological relatives or for individuals filing inheritance and social security benefit claims Y chromosomes are inherited intact, spontaneous mutations in the DNA sequence of the Y chrom are used to follow human migration patterns and historical lineages Y chrom genotyping has been used in studies designed to locate the geographical origin of all human beings all male relatives in a family, will share the same allele combination or profile > statistical significance of a Y-STR DNA match cannot be assessed by multiplying the likelihood ratios as in autosomal STRs; instead of allele frequency, haplotype frequencies are used Traditional STR loci are preferred for identity or relationship analyses Y-STRs are used to aid in special situations, e.g. in confirming sibship between males who share commonly occurring alleles (have a low likelihood ratio based on traditional STRs) Y-STRs have been used in forensic tests where evidence consists of a mixture of male and female DNA (semen, saliva, othe body fluid secretions, or finger nails scrappings e.g for evidence of rape, the female DNA may be in vast excess (more than 100-fold) compared to male DNA in the sample-autosomal STRs are not consistently informative under these circumstances using Y-specific primers, Y-STRs can be amplified by PCR from the male-female mixture, resulting an analyzable marker that has no female background > more accurate identification of the male donor

49. mtDNA and identity testing

before DNA isolation, the specimens are cleaned with detergent or for bone or teeth, by sanding to remove any possible sources of extraneous DNA adhering to the specimen the cleaned specimen is then ground in an extraction solution hair shafts yield mtDNA as do the fleshy pulp of teeth or bone the dentin layer of old tooth samples will also yield mtDNA mtDNA is isolated by organic extraction and amplified by PCR the PCR product is then purified and subjected to dideoxy sequencing a positive control of a known mitochondrial sequence is included with every run along with a reagent blank for PCR contamination and a negative control for contamination during the sequencing reaction if the negative or reagent blank controls yield sequences similar to the specimen sequence, the results are rejected both strands of the specimen PCR product must be sequenced occasionally, more than one mtDNA population is present in the same individual (heteroplasmy); in poiints of heteroplasmy, 2 DNA bases are observed at the same nt position; length heteroplasmy is a variation in the number of bases in tracts of like bases (homopolymeric tracts, e.g. CCCCCC); a length variant alone cannot be used to support an interpretation of exclusion if 2 or more nt differences occur between a reference and a test sample, the test sample can be excluded as originating from the reference or a maternally related person one nt difference between a reference and a test sample is interpreted as an inconclusive result if the test and reference samples show sequence concordance, then the test specimen cannot be excluded as coming from the same individual or maternal relative as the source of the reference sequence mtDNA profile of a test sample can also be searched in a population database population databases such as the mtDNA population dtatabase and the CODIS, are used to assess the weight of forensic evidence, based on the number of different mitochondrial sequences previously identified SWGDAM database contains mtDNA sequence information from more than 4100 unrelated individuals the probability of sequence concordance in 2 unrelated individuals is estimated at 0.003. the probability that 2 unrelated individuals will differ by a single base is 0.014 mtDNA analysis is used for lineage studies and to track population migrations same as Y-chroosome, there is no recombination between mitochnodria, and polymorphisms arise mostly through mutation the location and divergence of specific sequences in the HV regions of mitochondria are an historical record of the relatedness of populations becaise mtDNA are naturally amplified (hundreds per cell and 10 of circular genomes per mitochondria) and because of the nuclease- and damage-resistant circular nature of mtDNA, mtDNA typing has been a useful complement to other types of DNA identification challenging specimens of insufficient quantity or quality for nuclear DNA analysis may still yield useful information from mtDNA mtDNA analysis has been useful for the identification of missing persons in mass disasters or for typing ancient specimens mtDNA typing can also be applied to quality assurance issues, as described for STR typing of pathology specimens

32. Chimerism and mosaicism

chimera= an individual carrying 2 populations of cellsthat arose from different zygotes (of different genetic origin) mosaic=cells arising from the same zyote have undergone a genetic event, resulting in 2 clones of phenotypically different cells in the same individual

5. Steps of RE mapping

construct of an RE enzyme map of DNA region under investigation number and sizes of fragments of a test DNA region are compared with the number and sizes of fragments expected based on RE map polymorphisms are detected by observing fragment numbers and size different from the reference RE map detected by SB: DNA cut with RE, fragments are resolved by gel electrophoresis, blotted to a membrane, probes are then used to hybridize RFLP for detection

8. RFLP and parental testing

diploid organisms: chromosomal content is inherited half from each parent DNA polymorphism is also inherited this way one can infer a parent's contribution of alleles to a son or to a daughter from the combination of alleles in the child and those of other parent the RFLP fragment sizes of an individual are a combination of those from each parent in paternity testing, the alleles or fragment sizes of the offspring and the mother are analyzed. the remaining fragments (the ones that do not match the mother) have to come from the father. Alleged fathers are identified based on the ability to provide the remaining alleles (inclusion). A difference in just one allele may exclude paternity

29. First phase of allogenic transplantation

donor matching potential donors are tested for immunological compatibility it is performed by examining the human leukocyte antigen (HLA) locus to determine which donor would be most tolerated by the recipient immune system donors may be known or relating to the patient or anonymous unrelated contributor (matched unrelated donors [MUD] stem cells may also be acquired from donated umbilical cord blood

43. Quality assurance for surgical sections using STR

during processing of tissue specimens, microscopic fragments of tissue may persist in paraffin baths (floaters) tehse fragments can adhere to subsequent tissue sections, resulting in anomalous appearance of the tissue under the microscope; if a tusse is under question, STR identification can be used to confirm the origin of the tissue Procedure: suspected tissue must be carefully removed from the slide by microdissection; reference DNA isolated from the patient and DNA isolated from the tissue in question are subjected to multiplex PCR; the resulst are compared for matching alleles; if tissue in question originated from the patient, all alleles should match; one non-matching locus excludes the tissue in question as coming from the reference patient

6. RFLPS and genetics

each chromosome carries its polymorphisms offspring inherits a combination of the parental polymorphisms band patterns represent combination of RFLP inherited from each parent due to recombination and random assortment, each person as a unique set of RFLPS, half inherited from dad and half from mom every genotype will yield a descriptive band patterns over generations, mutation, intra- and inter-chromosomal recombination, gene conversion cause diversity a single locus (a gene or a region of DNA)will have several verions or allele human are diploid with 2 copies of every locus, i.e. each person has 2 alleles of each locuc if these alleles are same, the locus is homozygous if these alleles are different, the locus is heterozygous any 2 people can share the same alleles or have a different alles more closely related individuals are likely to share more alleles than unrelated persons 2 individuals can share both alleles at a single locus,but the chaces of 2 individuals, except identical twins, sharing the same alleles decrease 10x with each additionallocus tested > 2,000 RFLP loci RFLP protocols for human ID in North America use HaeIII. In Europe, HinfI is used NIST maintain reproducibility of RFLP process accross laboratories

9. Human identification using RFLP

first tool: ABO blood group antigens (the discrimination power is low; with only 4 possible groups, this method was only good for exclusion or elimination of a person as a source of biological material and was informatively only in 15-20% of cases) analysis of the polymorphic HLA loci could add a higher level of discrimination, with exclusion in 90% of cases testing both ABO and HLA could exclude a person in 97% of cases but still did not provide positive identification initial use of DNA as an identification tool relied on RFLP done with SB RFLP can arise from point mutations in the RE site, mutation that create a new RE site, and insertion or deletion of repeated sequences (tandem repeats). insertions or deletions of nts occurs frequently in repeated sequences in DNA tandem repeats are present in genomic DNA repeats of 8 or more nts are called variable number tandem repeats (VNTRs), or minisatellites VNTRs are large enough so that loss or gain of one repeat can be resolved by gel electrophoresis of a RE digest frequent cutters: HaeIII (recognition site: GGCC) or HinfI (recognition site: GANTC), generated fragments that are small enough to resolve and give an informative pattern by SB first human DNA profiling system 1985: UKFSS using Alec Jeffrey's SB multiple locus MLP-RFLP system. it uses 3-5 probes to analyze 3-5 loci on the same blot single locus probe (SLP) SYSTEM 1990: used in Europe and North America; analysis of 1 locus at a time yield simpler patterns, which were much easier to interpret, esp. in cases where specimens may contain DNA from more than one individual

2. Human sequence polymorphisms

human sequence polymorphisms affect many base pairs large blocks of repeated sequences may inverted, deleted, or duplicated from one individual to another long interspersed nucleotide sequences (LINES)=highly repeated sequences , 6-8 kbp, that contain RNA polymerase promoter s and open reading frames related RT'ase of retroviruses>500,000 of these LINE-1 (L10 elements (15% of human genome) short interspersed nucleotide sequences (SINES)= scattered over genome, 0.3 kbp, > 1,000,000 copies per genome SINES includes Alu elements (harboring recognition site of Alu enzyme LINES ad SINES are also called mobile elements or transposable elements, they are copied and spread by recombination and reverse transcription, they may produce pseudogenes (intronless, non fx copies of human genome short blocks of repeated sequences also undergo expansion or shrinkage through generations: they are STRs (short-tandem repeats) and VNTRs (variable number tandem repeats) SNPs, larger sequence variants, and tandem repeats can be detected by RFLP SNPS, VNTRs, STRs, and RFLPs are routinely used in clinical labs

31. Monitoring of engraftment

in the first 90 days of engraftment it requires distinguishing between donor cells and recipient cells red blood cell phenotyping, immunoglobin allotyping, HLA typing, and FISH, all have been used for this purpose

1. Polymorphisms

large size of human genome: 2 billion bp 2% of human genome codes for genes probability of polymorphism DNA is high genome sequences diifer by 1 nt for every 1000-1500 bases single nucleotide polymorphisms (SNPs)=single nt differences in human genomes polymorphism are more frequent in some areas of genome than the others HLA(human leukocyte antigen) locus is a highly polymorphic region of human DNA HLA genes codes for peptides that establish self-identity of the immune system similarity or compatibility between immune system of transplant recipients and potential donors can be determined by comparing DNA sequences HLA typing may also be used for exclusion in human identification tests

42. Linkage analysis

many STRs in the genome are known they can be used to map genes, esp. those genes associated with diseases 3 approaches are used to map genes: family history, population studies, and sibling analyses family history and analysis of generations of a single family for the presence of a particular STR allele in affected individuals is one way to show association; family members are tested for several STRs, and the alleles of affected and non-affected members are compared; if a particular allele of a particular locus is always present in affected family members, that locus must be closely linked to the gene responsible for the phenotype in those individuals (linkage disequilibrium); if the linkage is closed enough to the gene (no recombination between the STR and the disease gene), the STR may serve as a convenient target for disease testing; in this later case, the presence of the indicator STR allele serves as a genetic marker for the disease Another approach for linkage analysis is to look for gene associations in large numbers of unrelated individuals in population studies; close linkage to specific STR alleles support the genetic proximity of the disease gene with STR Sibling studies is another type of linkage analysis; monozygotic (identical) and dizygotic (fraternal) twins serve as controls for genetic and environmental studies; monozygotic twins will always have the same genetic alleles, including diseases genes; there should be a 100% recurrence risk (likelihood) that if one twin has a genetic disease, the other twin has it, and both should have the same lnked STR alleles; fraternal twins have the same likelihood of sharing a gene allele as any sibling pair

24. Matching with Y-STRs

matching probability data from Y-STR data are determined differently than for the autosomal STR haplotype diversity (HD) is calculated from the frequency of a given haplotype in a tested population the probability of 2 random males sharing the same haplotype is estimated at 1-HD; i.e., if the haplotype diversity is high, the probability of 2 random males in the population having the same haplotype is low discriminatory capacity (DC) is another measure of profile uniqueness DC is determined by the number of different haplotypes seen in the tested population and the total number of samples in the population DC expresses % of male in a population who can be identified by a given haplotype DC is increased by increasing the number of loci defining a haplotype e.g. the loci tested in Y-Plex 6 system can distinguish 82% of African males. Using 22 loci raises the DC to almost 99% b/c thereis no recombination between loci on the Y chrom, the product rule cannot be applied results of a Y typing might be reported accompanied by the number of observations or frequency of the analyzed haplotype in a database of adequate size e.g. a hapolytpe containing 17 alleles of DYS390 occurs in only 23% of men in a database of 12,400.if that same haplotype contains 21 alleles of DYS446, only 6% of men will have haplotype containing both the DYS390 17 and DYS446 21 alleles. If the 11 alleles of DYS445 and 15 alleles of DYS458 are also present, only 1 out of 12,400 men in the population has a haplotype containing all four alleles the unique of this haplotype is strong evidence that a match is not a random coincidence however, even a 99%DC, the matching probability is orders of magnitude lower than that of autosomal STR Y-chromosome haplotype can be used to exclude patertinity any alleles that differ between the male child and the alleged father are strong evidence for nonpaternity if a Y halotype is shared between a child and alleged father, a paternity index is calculated in a manner similar to that of autosomal STR analysis e.g. suppose 6 Y-STR alleles are tested and match between the alleged father and child. if the haplotype has not been observed before in the population, the occurrence of that haplotype in the population database is 0/1200, and the haplotype frequency will be 1/1200 or 0.0008333. the paternity index (PI) is the probability that a man with that haplotype is could produce one sperm carrying the haplotype. PI is then 1/0.00083333 = 1200. With a prior probability is 0.5, the PI is (1200 x 0.5)/[(1200 x 0.5) + 0.5] or 99.9% this result, however, does not exclude patrilineal relatives of the alleged father Y-STR as a maker loci for Y-chromosome or surname tests are used to determine ancestry e.g. a group of males of a strictly male descent line (having the same last name or surname) is expected to be related to a common male ancestor. They should all share the same Y-chromosome alleles (except for mutation which is minimal, given 1 mutation over 20 generations) the Y-chromosome haplotype does not provide information about the degree of relatedness, just inclusion or exclusion from the family an analysis to find a most recent common ancestor (MRCA) is possible using a combination of researched family histories, Y-STR test results, and statistical formulas for mutation frequencies

33. DNA typing

method of choice for engraftment monitoring all individuals (except identical twins) have unique DNA polymorphisms donor cells are monitored by following donor polymorphisms in the recipient blood or bm RFLP can effectively distinguish donor and recipient cells but detection of RFLP requires the use of SB (labor-intensive and has limited sensitivity for this application) small VNTRs and STRs are easily detected by PCR amplification; they are prefferrred b/c of the increased rapidity and the 0.5-1% sensitivity achievable with PCR; sensitivity can be raised to 0.01 using Y-STR Y-STR is limited to those transplants from sex-mismatched donors ( from a female donor to a male recipient)

23. Y-STR loci

minimal haplotype: a set of Y-STR loci established by the European typing community; include DYS19, DYS389i, DYS389II, DYS390I, DYS391, DYS392, DYS 393,and DYS385 extended haplotype: includes mini haplotype + highly polymorphic dinucleotide repeats YCAII Y-STRs have microvariant alleles containing repeat sequence differences Y-STR kits: PowePlex system, 12 Y loci (Promega), AmpliSTR Y-filer, 17 loci (Life Technology/ABI) and the ReliaGene, Y-Plex 6-contains 6 Y loci

48. mtDNA sequence

mtDNA sequence are divided into 2 components: forensic and public the forensic component consists of anonymous population profiles and is used to assess the extend of certainty of mtDNA identification in forensic casework all forensic profiles include a sequence region in HVI (nt. 16024-16384) and a sequence region in HVII (nt. 53-372) these data are searched through the CODIS in open case files and missing person cases ~610 bp, including the hypervariable regions of mtDNA, are routinely sequenced for forensic analysis a transition from A to G at position 263 would be recorded as 263G the public data consist of mtDNA sequence data from the scientific literature and the GenBank and European Molecular Biology Laboratory databases public database provides information on worldwide population groups not contained within the forensic data and can be used for investigative purpose all maternal relatives share mtDNA sequencesand daughter will exactly match in the hypervariable region in the abscence of mutaion; thus, the use of mtDNA polymorphism is for exclusion there is an average of 8.5 nt differencesbetween mtDNA sequences of unreated individuals in the hypervariable region

7. Genetic mapping with RFLPS

polymorphisms are inherited in a Mendelian fashion locations of many polymorphism are known polymorphisms can be used as landmarks or markers in the genome to determine the location of other genes one can confirm that a disease has a genetic component by demonstrating a close genetic association or linkage to a known marker the more frequently a particular polymorphism is present in persons with a disease phenotype, the more likely the affected gene is located close to the polymorphism (linkage mapping principle) e.g. a RFLP for inherited breast cancer has a location 17q21 and always present in families with high incidence of breast and ovarian cancers. Therefore, the BRCA1 gene is mapped to this position on the long arm of chromosome 17

39. Cell separation techniques

positive or negative selection techniques may be used to test specific cell lineage e.g. analysis of T-cell fraction separately is used to monitor graft-versus-tumor potential; T cells may comprise 10% of peripheral blood leukocytes and 3% of bm cells following allogenic transplantation; analysis of unfractionated blood and esp. bm where all other cell lineages are 100% could miss spli chimerism in the T-cell fraction; T-lineage-specific chimerism will therefore increase the sensitivity of the engraftment analysis, particularly after myeloablative and immunoablative pretransplant treatments T cells are separated from whole blood using magnetized polymer particles (beads): e.g. MicroBeads (MicroBeads AS), DynaBeads (Dynal), EasySep (Stem Cell Technologies), attached to pan-T antibodies (anti-CD3) To isolate T cells using beads, WBC isolated by density gradient centrifugation are mixed with the beads in saline or PBS and incubated to allow the Ab on the beads to bind to CD3 antigen on the T-cell surface; with the beads T cells are immobilized by a magnet that is applied to the outside of the tube and the supernatant containing non-T cells is decanted; after another saline wash, T cells are collected and lysed for DNA isolation Automated cell sorter systems such as the AutoMACS separator (Milteni Biotec) may also be used for this purpose; with the positive selection program, it is caable of isolating up to 2 x E8 pure T cells per separation; unwanted cells can be removed with the depletion program

12. STR analysis

smaller STRs are efficiently amplified by PCR long, intact DNA fragments are not required to detect the STR products, therefore, degraded or otherwise less-than-optimal specimens are potentially informative amount of specimen required for STR analysis is 10 ng STR alleles are identified by PCR product size primers are designed to produce amplicons of 100-400 bp sizes of the PCR products are influenced by the number of embedded repeats one of the primer is labeled with a fluo marker and PCR product can be analyzed in fluorescent-detection system silver-stained gels may be used capillary electrophoresis with fluorescent dyes is preferred

40. Special situations

specimens may be received in thelab after engraftment without pre-engraftment information. In this case, the blood and bm of recipient is not acceptable for determination of recipient-specific alleles because the alleles present my represent donor, recipient, or both donor and recipient the specimen can be processed using the amelogenin locus or Y-STR markers if the donor and recipient are of different sexes, preferably female donor and male recipient another option is to use alternate source of recipient DNA such as biccal cells, skin biopsy sample, or stored specimens or DNA from previous testing Because of the nature of lymphocyte migration, skin and buccal cells may also have donor alleles due to the presence of donor leukocytes in these tissues The best approach is to ensure that informative analysis of donor and recipient are part of the pre-transplant agenda

15. DNA genotyping or profiling

test DNA is mixed with primer pair, buffer, and polymerase to amplify the test loci a control DNA standard is also amplified each PCR product is combined with allelic ladders (sets of fragments representing all possible alleles of a repeat locus) and internal size standards (MW markers) in formamide for electrophoresis analysis software will size and identify the alleles in contrast to RFLPs and VNTRs, STRs are discrete allele systems, in which a finite number of alleles are defined by the number of repeat units in the tandem repeat commercial systems have labeled primers for 1 to more than16 loci commercial primer sets are designed with "stuffer" sequences to modify the size of the PCR products so that, the range of alleles for 4-5 loci can be resolved by gel or capillary electrophoresis the product size for a given allele will not always be the same with primers from different commercial sources allelic ladders in the reagent kits allow accurate identification of the sample alleles test DNA amplicons, allelic ladders, and size standards for multiple loci are run simultaneously through each capillary genotyping software: GeneMapper (ABI), STaR Call, and FMBIO Analysis Software (Hitachi) provide automated resolution of fluorescent dye colors and genotyping by comparison with the size standards and the allelic ladder STR match is made by comparing profiles followed by probability calculation AmpliType HLA DQa Forensic DNA amplification and Typing kit (Promega) have been used in conjunction with PolyMarker 9PM) system to generate highly discriminatory allele frequencies, e.g. the chance of a set of alleles occurring in 2 unrelated individuals at random will be: 1 in 10E6 to 7 x 10E8 in Caucasians 1 in 3 x 10E6 to 3 x 10E8 in African Americans