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Polymorphisms Polymorphism A DNA polymorphism is a sequence difference compared to a reference standard that is present in at least 1%–2% of a population. Polymorphisms can be single bases or thousands of bases. Polymorphisms may or may not have phenotypic effects Polymorphic DNA Sequences Polymorphisms are found throughout the genome. If the location of a polymorphic sequence is known, it can serve as a landmark or marker for locating other genes or genetics regions. Each polymorphic marker has different versions, or alleles. Types of Polymorphic DNA Sequences RFLP: restriction fragment length polymorphisms VNTR: variable number tandem repeats (8 to >50 base pairs) STR: short tandem repeats (1–8 base pairs) SNP: single-nucleotide polymorphisms Types of Polymorphisms Single nucleotide polymorphisms (SNPs) A Single Nucleotide Polymorphism, or SNP (pronounced "snip"), is a small genetic change, or variation, that can occur within a person's DNA sequence. Common SNPs are defined as >1% in at least one population Rare SNPs are hard to identify and validate But, it is estimated that there are a large number per individual Some areas more variable than others (HLA) Restriction Fragment Length Polymorphisms Restriction fragment sizes are altered by changes in or between enzyme recognition sites. GTCCAGTCTAGC GAATTC GTGGCAAAGGCT CAGGTCAGATCG CTTAAG CACCGTTTCCGA GTCCAGTCTAGC GAA ATC CG TGGC CAAGGCT CAGGTCAGATCG CTTTAG GC ACCG GTTCCGA Point mutations GTCCAGTCTAGC GA AGCGA ATTC GTGGC AAAGGCT CAGGTCAGATCG CTTCGCT TAAG CACCG TTTCCGA Insertion GTTCTAGC GAATTC GTGGC AAA GGCT GAATTC GTGG TCAGATCG CTTAAG CACCG TTTCCGA CTTAAG CACC GTTCTAGC GAATTC GTGGC AAAAAA GGCT GAATTC GTGG TCAGATCG CTTAAG CACCG TTTTT TCCGA CTTAAG CACC Fragment insertion (or deletion) RFLP RFLP (often pronounced "rif lip”) is used to follow a particular sequence of DNA as it is passed on to other cells. RFLPs can be used in paternity cases or criminal cases to determine the source of a DNA sample. RFLPs can be used determine the disease status of an individual. RFLPs can be used to measure recombination rates which can lead to a genetic map with the distance between RFLP loci measured in centiMorgans. RFLP An RFLP is a sequence of DNA that has a restriction site on each end with a "target" sequence in between. A target sequence is any segment of DNA that bind to a probe by forming complementary base pairs. A probe is a sequence of single-stranded DNA that has been tagged with radioactivity or an enzyme so that the probe can be detected. When a probe base pairs to its target, the investigator can detect this binding and know where the target sequence is since the probe is detectable. RFLP RFLP produces a series of bands when a Southern blot is performed with a particular combination of restriction enzyme and probe sequence RFLP Recall: EcoR I binds to its recognition sequence GAATTC and cuts the double-stranded DNA as shown EcoRI -GAATTC-CTTAAG- In the segment of DNA shown below, you can see the elements of an RFLP; a target sequence flanked by a pair of restriction sites. When this segment of DNA is cut by EcoR I, three restriction fragments are produced, but only one contains the target sequence which can be bound by the complementary probe sequence (purple). Restriction Fragment Length Polymorphisms RFLP genotypes are inherited. For each locus, one allele is inherited from each parent. Father Locus 1 2 Mother Locus 1 2 Southern blot band patterns Parents 1 Child Locus 2 Let's look at two people and the segments of DNA they carry that contain this RFLP (for clarity, we will only see one of the two stands of DNA). Since Jack and Jill are both diploid organisms, they have two copies of this RFLP. When we examine one copy from Jack and one copy from Jill, we see that they are identical: Jack 1: -GAATTC---(8.2 kb)--- GCATGCATGCATGCATGCAT---(4.2 kb)---GAATTC Jill 1: -GAATTC---(8.2 kb)--GCATGCATGCATGCATGCAT---(4.2 kb)---GAATTC- When we examine their second copies of this RFLP, we see that they are not identical. Jack 2 lacks an EcoR I restriction site that Jill has 1.2 kb upstream of the target sequence (difference in italics). Jack 2: -GAATTC--(1.8 kb)-CCCTTT--(1.2 kb)-- GCATGCATGCATGCATGCAT--(1.3 kb)-GAATTC- Jill 2: -GAATTC--(1.8 kb)-GAATTC--(1.2 kb)-GCATGCATGCATGCATGCAT--(1.3 kb)-GAATTC- Therefore, when Jack and Jill have their DNA subject to RFLP analysis, they will have one band in common and one band that does not match the other's in molecular weight: Parentage Testing by RFLP Which alleged father’s genotype has the paternal alleles? AF2 Mother AF 1 Locus 1 2 Locus 1 2 Child Locus 1 2 Locus 1 2 Evidence Testing by RFLP Which suspect—S1 or S2—was at the crime scene? (V = victim, E = crime scene evidence, M = molecular weight standard) M S1 S2 V E M Locus 1 M S1 S2 V E M Locus 2 M S1 S2 V E M Locus 3 Short Tandem Repeat Polymorphisms (STR) STRs are repeats of nucleotide sequences. AAAAAA…: mononucleotide ATATAT…: dinucleotide TAGTAGTAG…: trinucleotide TAGTTAGTTAGT…: tetranucleotide TAGGCTAGGCTAGGC…: pentanucleotide Different alleles contain different numbers of repeats. TTCTTCTTCTTC : four-repeat allele TTCTTCTTCTTCTTC: five-repeat allele Short Tandem Repeat Polymorphisms STR alleles can be analyzed by fragment size (Southern blot). Restriction site One repeat unit Allele M 1 2 M Allele 1 GTTCTAGC GGCC GTGGC AGCTAGCTAGCTAGCT GCTG GGCC GTGG CAAGATCG CCGG CACCG TCGATCGATCGATCGA CGAC CCGG CACC tandem repeat Allele 2 GTTCTAGC GGCC GTGGC AGCTAGCTAGCT GCTG GGCC GTGG CAAGATCG CCGG CACCG TCGATCGATCGA CGAC CCGG CACC Short Tandem Repeat Polymorphisms STR alleles can also be analyzed by amplicon size (PCR). Primers Allele 1 ....TCATTCATT CATT CATT CATTCATT CAT.... ....AGTAAGTAAGTAAGTAAGTAAGTAAGTA.... Allele 2 ....TCAT TCATT CATTCATT CATT CATTCATTCAT.... ....AGTAAGTAAGTAAGTAAGTAAGTAAGTAAGTA.... PCR products: Allele 1 187 bp (7 repeats) Allele 2 191 bp (8 repeats) (Genotype) 7/8 Short Tandem Repeat Polymorphisms Allelic ladders are standards representing all alleles observed in a population. 11 repeats (Allelic ladder) 5 repeats Genotype: 7,9 Genotype: 6,8 Short Tandem Repeat Polymorphisms Multiple loci are genotyped in the same reaction using multiplex PCR. Allelic ladders must not overlap in the same reaction. Short Tandem Repeat Polymorphisms by Multiplex PCR FGA PentaE TPOX D18S51 D2S11 D8S1179 THO1 vWA D3S1358 Amelogenin Locus, HUMAMEL The amelogenin locus is not an STR. The HUMAMEL gene codes for amelogenin-like protein. The gene is located at Xp22.1–22.3 and Y. X allele = 212 bp Y allele = 218 bp Females (X, X): homozygous Males (X, Y): heterozygous Analysis of Short Tandem Repeat Polymorphisms by PCR STR genotypes are analyzed using gel or capillary gel electrophoresis. 11 repeats 5 repeats 11 repeats 5 repeats (Allelic ladder) Genotype: 7,9 STR-PCR STR genotypes are inherited. Child’s alleles Mother’s alleles Father’s alleles One allele is inherited from each parent. Parentage Testing by STR-PCR Which alleged father’s genotype has the paternal alleles? Locus D3S1358 vWA FGA TH01 TPOX CSF1PO D5S818 D13S317 Child 16/17 14/18 21/24 6 10/11 11/12 11/13 9/12 Mother 16 16/18 20/21 6/9.3 10/11 12 10/11 9 1 17 14/15 24 6/9 8/11 11 13 12/13 2 17 16/17 24 6/7 8/9 11/13 9/13 11/12 Evidence Testing by STR-PCR Which suspect—S1 or S2—was at the crime scene? (V = victim, E = crime scene evidence, AL = allelic ladder) AL S1 S2 V E M AL S1 S2 V E M AL S1 S2 V E M Locus 1 Locus 2 Locus 3 Short Tandem Repeat Polymorphisms: Y-STR The Y chromosome is inherited in a block without recombination. STR on the Y chromosome are inherited paternally as a haplotype. Y haplotypes are used for exclusion and paternal lineage analysis. Short Tandem Repeat Polymorphisms: miniSTR Mini-STRs are STRs on smaller amplicons. Recommended for degraded specimens Used to identify remains from mass graves and disaster areas MHC Major histocompatibility complex (MHC) is a cell surface molecule encoded by a large gene family in all vertebrates. MHC molecules mediate interactions of leukocytes. MHC determines compatibility of donors for organ transplant as well as one's susceptibility to an autoimmune disease via crossreacting immunization. In humans, MHC is also called human leukocyte antigen (HLA). Protein molecules—either of the host's own phenotype or of other biologic entities—are continually synthesized and degraded in a cell. Occurring on the cell surface, each MHC molecule displays a molecular fraction, called an epitope. The presented antigen can be either self or nonself. The MHC gene family is divided into three subgroups—class I, class II, and class III. We will discuss these later….. SNPs & Function: We know so little.. Majority are “silent”: No known functional change Alter gene expression/regulation Promoter/enhancer/silencer mRNA stability Small RNAs Alter function of gene product Change sequence of protein Large and Small Scale Polymorphisms Copy Number of Polymorphisms Regional “repeat” of sequence 10s to 100s kb of sequence Estimate of >10% of human genome Multi-copy in many individuals Duplicons 90-100% similarity for >1 kb 5-10% of genome (5% exons elsewhere) Multi-copy (high N) in all individuals Copy Number Variation Across the Genome Frequency of CNVs Most are uncommon (<5%) Familial vs Unrelated Studies Associated with Disease OPN1LW Red/green colorblind CCL3L1 Reduced HIV Infection CYP2A6 Altered nicotine metabolism VKORC1 Warfarin metabolism Engraftment Testing Using STR Allogeneic bone marrow transplants are monitored using STR. Autologous transplant Recipient receives his or her own purged cells. Allogeneic transplant Recipient receives donor cells. A recipient with donor marrow is a chimera. Engraftment Testing Using STR There are two parts to chimerism testing: pretransplant informative analysis and post-transplant engraftment analysis. Before transplant Donor Recipient After transplant Complete (full chimerism) Mixed Graft failure chimerism Engraftment Testing Using STR: Informative Analysis STR are scanned to find informative loci (donor alleles differ from recipient alleles). Which loci are informative? Locus: M 1 D 2 R D 3 R 4 D R 5 D R D R Engraftment Testing Using STR: Informative Analysis There are different degrees of informativity. With the most informative loci, recipient bands or peaks do not overlap stutter in donor bands or peaks. Stutter is a technical artifact of the PCR reaction in which a minor product of n-1 repeat units is produced. Examples of Informative Loci (Type 5) [Thiede et al. Leukemia 2004;18:248] Recipient Stutter Donor Recipient Donor Examples of Non-informative Loci (Type 1) Recipient Donor Engraftment Testing Using STR: Informative Analysis Which loci are informative? vWA TH01 Amel TPOX CSF1PO Engraftment Testing Using STR: Engraftment Analysis Using informative loci, peak areas are determined in fluorescence units or from densitometry scans of gel bands. A(R) + A(D) A(R) A(D) area under recipient-specific peaks A(R) = A(D) = area under donor-specific peaks Engraftment Testing Using STR: Engraftment Analysis Formula for calculation of % recipient or % donor (no shared alleles). % Recipient DNA = % Donor DNA = A(R) A(R) + A(D) A(D) A(R) + A(D) × 100 × 100 Engraftment Testing Using STR: Engraftment Analysis Calculate % recipient DNA in post-1 and post-2: Use the area under these peaks to calculate percentages. Engraftment Analysis of Cellular Subsets Cell subsets (T cells, granulocytes, NK cells, etc.) engraft with different kinetics. Analysis of cellular subsets provides a more detailed description of the engrafting cell population. Analysis of cellular subsets also increases the sensitivity of the engraftment assay. Engraftment Analysis of Cellular Subsets T cells (CD3), NK cells (CD56), granulocytes, myeloid cells (CD13, CD33), myelomonocytic cells (CD14), B cells (CD19), stem cells (CD34) Methods Flow cytometric sorting Immunomagnetic cell sorting Immunohistochemistry + XY FISH Engraftment Analysis of Cellular Subsets Detection of different levels of engraftment in cellular subsets is split chimerism. R R = recipient alleles D = donor alleles T = T-cell subset (mostly recipient) G = granulocyte subset (mostly donor) D T G Single Nucleotide Polymorphisms (SNPs) Single-nucleotide differences between DNA sequences. One SNP occurs approximately every 1250 base pairs in human DNA. SNPs are detected by sequencing, melt curve analysis, or other methods. 99% have no biological effect; 60,000 are within genes. SNP Detection by Sequencing T/T 5′ AGTCTG T/A 5′ AG(T/A)CTG A/A 5′ AGACTG SNP Haplotypes SNPs are inherited in blocks or haplotypes. haplotype ~10,000 bp Applications of SNP Analysis SNPs can be used for mapping genes, human identification, chimerism analysis, and many other applications. The Human Haplotype Mapping (HapMap) Project is aimed at identifying SNP haplotypes throughout the human genome. Mitochondrial DNA Polymorphisms Sequence differences in the hypervariable regions (HV) of the mitochondrial genome HV 1 (342 bp) PH1 PH2 HV 2 (268 bp) PL Mitochondrial genome 16, 600 bp Mitochondrial DNA Polymorphisms Mitochondria are maternally inherited. There are an average of 8.5 base differences in the mitochondrial HV sequences of unrelated individuals. All maternal relatives will have the same mitochondrial sequences. Mitochondrial typing can be used for legal exclusion of individuals or confirmation of maternal lineage. Summary Four types of polymorphisms are used for a variety of purposes in the laboratory: RFLP, VNTR, STR, and SNP. Polymorphisms are used for human identification and parentage testing. Y-STR haplotypes are paternally inherited; maternal relatives have the same mitochondrial DNA alleles. Polymorphisms are used to measure engraftment after allogeneic bone marrow transplants. Summary Single-nucleotide polymorphisms are detected by sequencing, melt curve analysis, or other methods. SNPs can be used for the same applications as other polymorphisms. Mitochondrial DNA typing is performed by sequencing the mitochondrial HV regions. Mitochondrial types are maternally inherited.