Non-autosomal markers - created from Mind Map

Page 1

chromosomes with no role in sex determination<br><br><ul><li>Autosomes • Routine DNA profiling techniques… » Autosomal STR profiling </li></ul>

Sex determining chromosomes<br><br><ul><li>Obviously contain DNA </li></ul>

Mitochondria<br><br><ul><li> Sub-cellular organism with extrachromosomal DNA (mtDNA) </li></ul>

Non-Autosomal DNA profiling<br><br><ul><li>Profiling variation on X and Y chromosomes, or from the mtDNA genome </li><li>Used for Routine DNA profiling techniques…STRs </li></ul>

mtDNA profiling

For a match...<br><br><ul><li>Need database context</li><li>The mtDNA type is searched for in a database of mtDNA Current FBI version contains:

1332 African/African origin 686 Hispanic 326 Native American 821 Asian 1674 Caucasian </li></ul>

IF type previously observed<br><br><ul><li>95% confidence interval</li></ul>

type never observed<br><br><ul><li>95% upper bound</li></ul>

Presenting courtroom statistics

Mutation

Heteroplasmy<br><br><ul><li>Can be sequence or length based • Can occur in three different ways: » A single tissue type from an individual may have more than one mtDNA type » Different tissues from the same person may exhibit different mtDNA types » Some tissues may have more than one mtDNA type but another tissue from the same person has only one mtDNA type Buckleton </li><li>obably exists in all individuals » Most often below a level of detection for routine sequencing techniques used » Variant needs to be ~20% of non-mutated base » Detection limits vary depending on the sequencing chemistry used • Controversial report of increased heteroplasmy in hair roots [1] » Use of nested PCR [2] and contamination [3] raised as contrary source for observed results • Some consensus regarding location of mutation and heteroplasmy “hot spots</li><li>Can be sequence or length heteroplasmy • Sequence – presence of 2 nucleotides at a single position shown as an overlapping sequence electropherogram </li></ul>

Romanov<br><br><ul><li>STR analysis compared presence of family group in grave corresponding mtDNA sequences from three putative grand suchessrs to Tsarina supported family </li></ul>

Y chromosome analysis<br><br><ul><li>Is our smallest chromosome • Contains only 2% of the entire genome • Is approximately 60Mb in length • Is non-recombining for approximately 95% of its length » Known as non-recombining region (NRY) or male specific region (MSY) • Recombination only occurs at the distal ends of the long and short arms » Known as the pseudoautosomal regions 1 and 2 (or PAR-1 and PAR-2)</li></ul>

Summary<br><br><ul><li>Refining applications for non-autosomal DNA profiling has been of considerable value to the field of forensic biology » The investigative relevance of the techniques » The capability of casework laboratories has also increased • </li><li>» Involved in anthropological and evolutionary research for several years » Able to provide a result when gDNA markers fail » Suitable for otherwise difficult samples (fingernails, bone, telogenic hairs) » Becoming more relevant given modern context… » For example; Terrorism (DVI) and Refugees (Spanish Phoenix Program) </li></ul>

Role of mitochondria<br><br><ul><li> Are the remnants of symbiotic proteobacteria (e.g. Rickettsia ) that were ingested by a eukaryotic cell with no cell wall • Contain their own DNA which, in common with bacteria, is a double stranded closed circular molecule • Some of the ancestral bacteria genes have transferred to the nuclear DNA, but ~16,569 bases remain within the mitochondrion • The mtDNA molecule contains coding and non-coding areas • The coding regions code for the 37 remaining genes of the mitochondria… » Comprising 22 transfer RNAs, 2 ribosomal RNAs, and 13 proteins </li></ul>

Up to 10,000 mitochondria in every cell<br><br><ul><li>~10,000 copies of mtDNA » Compared to one copy of gDNA </li></ul>

Is a separate organelle…<br><br><ul><li> protected by additional membrane </li></ul>

mtDNA is covalently closed<br><br><ul><li>Affording greater stability </li></ul>

Nomenclature<br><br><ul><li>Complete mtDNA sequence… • First published by Anderson et al., in 1981 [1] » “Anderson sequence” or “Cambridge Reference Sequence” » Does not originate from a single individual » Contains the rarer variant at some positions, notably 263 and 315.1 » Contains several sections of bovine and HeLa sequence » Preferred overall to describe the control region, due to widespread acceptance • Starts at arbitrary position near origin of H-strand replication » Each base given a consecutive number from 1 to 16569 • Any mtDNA sequence can be described as a list of differences from the “Anderson sequence”

[</li><li>Of the two bases on each of the complementary strands it is the base on the cytosine-rich light strand (L-strand) that is quoted » For example in the Anderson sequence at position 280 the L-strand is C » If the sequence to be reported differs at this position then it is reported

[</li></ul>

greatest forensic interest<br><br><ul><li>control region</li><li> Also termed the D-loop (displacement loop) » 1125 bp section flanking the origin of replication. • It is non-coding… » Although contains the light (L-) and heavy (H-) strand promoters and other transcription factors and regulatory elements </li><li>Two portions of the control region display significant variability • Termed hypervariable regions 1 and 2 (HV I and HV II respectively) » HV I extends from position 16024 to approximately 16365 » HV II extends from approximately position 73 to approximately 340 </li></ul>

disadvantages to sequencing<br><br><ul><li>expensive, primarly due to labour

also high error possibilities with more data to review and most info not used</li></ul>

Maternal inheritance<br><br><ul><li>• Demonstrated in humans through following studies of multiple extended lineages » rare evidence of paternal contribution ? • The emerging consensus is that recombination in humans is either minimal or does not occur • For practical purposes in forensic identification cases maternal inheritance, alone, is usually assumed. </li></ul>

sanger method<br><br><ul><li>based on the selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication.</li></ul>

results

Specialist applications... Forensic

control region<br><br><ul><li>1122 nucleotide positions\

only 610 bases examined</li></ul>

coding region<br><br><ul><li>15446 nucleotide positions

challenged with typing widley spaced SNps and multiplex PCR required

polymorphisms may have medical significance</li></ul>

point heteroplasmy<br><br><ul><li>Several options… » Alternatively, the designation T~C could be used » If one base is in higher proportion than other, notation such as C&amp;gt;T may be used » If the position remains ambiguous then it will be labelled (N) or the appropriate IUB designation may be used [R=(A or G), Y=(C or T), K=(G or T), M=(A or C), B=(C,G, or T), D=(A,G, or T), H=(A,C, or T), V=(A,C, or G), S=(G or C), W=(A or T)] </li></ul>

length variants<br><br><ul><li>Insertions » Reported at the insertion position with a &quot;.1&quot; after the lower numbered base » For example 245.1A means an adenosine base was inserted after base 245 </li><li> Particularly common in poly(C) stretches » Due to the homopolymeric stretch, the exact location of the insertion is ambiguous » The insertion is labelled as occurring at the upper end of the homopolymeric tract » For example, an insertion in the poly(C) stretch between positions 302 and 310 would be designated 309.1C; two insertions here would be listed as 309.1C and 309.2C

Buckleton </li></ul>

deletions<br><br><ul><li>Deletions… » Reported by nominating the deleted base with a “d” » For example if the base between 245 and 247 was deleted, would list as “246d”</li></ul>

highly degraded samples<br><br><ul><li>Skeletal remains</li></ul>

samples with very little gradient<br><br><ul><li> Hair shafts </li></ul>

samples with no reference<br><br><ul><li>Establish identity via extrapolation of maternal lineage</li></ul>

other<br><br><ul><li>Human evolution and population-specific diversity • Population growth and migration • Historical investigations </li></ul>

number of occurences in database<br><br><ul><li>for example, “The type in question has been observed 5 times in 34,316 individuals” (= 1 person in 6,863) </li></ul>

confidence interval calculated around frequency estimate<br><br><ul><li>for example, “This frequency is unlikely to be more common in the overall population than at least 1 in 3,657.” (there is only a 2 ½ % chance that it is more common than that.)

5 </li></ul>

mtDNa higher mutation rate<br><br><ul><li>Up to 20 times higher than microsatellites » Some variance in observed and expected rates and between short-course and phylogentic studies [1]</li></ul>

common<br><br><ul><li>Single base substitutions are most common • Particularly in homopolymeric stretches » Polycytosine regions (“poly(C) stretches”) between positions 302 and 310 and positions 16183 and 1619</li></ul>

forensic significance<br><br><ul><li>Evidential (crime stain) and reference samples may contain differences even when both are from the same individual or lineage [</li></ul>

mutations<br><br><ul><li>Passed between generations in varying ratios • Segregate during development and later life • This results in mixtures of mtDNA molecules that differ from each other at one or more bases…this is known as “heteroplasmy” </li></ul>

mutation in progress

Jesse James<br><br><ul><li>matched sister</li></ul>

Grand Duke showed heteroplsmy<br><br><ul><li>both borthers must have inherited two types of mitochondria from thei mother</li></ul>

cons<br><br><ul><li>Loci are not independant of one another and rare random match probabilities cannot be generated with product rule and must use haplotypes generated with product rule</li><li>paternal lineages possess the same Y-STR haplotype and thus fathers sons, brothers and uncles cannot eb distinguished</li></ul>

Y chromosome degrading, lots lots of genes and geneticists predict the eradication of the Y chromosome in 9 million years

human Y<br><br><ul><li>Has 78 protein coding sequences in the MSY » These encode 27 distinct proteins » Proportionally lower than the autosomes • Has an array of different polymorphisms suitable for PCR-based analysis including… » Single nucleotide polymorphisms (SNP’s), microsatellites and minisatellites • For similar reasons to autosomal markers STR’s are preferred for forensic purposes » Roewer et al. [1] reported the first STR on the Y chromosome now known as DYS19 </li><li>The study of human evolution and migration • The study of ethnic populations and their genetic diversity • The resolution of historical investigations </li></ul>

Y-STR<br><br><ul><li> proven valuable for forensic work • When there is a low level of male DNA in comparison to the amount of female DNA present in a sample • When a study of family relationships are needed » Patrilineal inheritance (with no recombination</li><li> A variety of commercial Y-STR multiplexes available » Promega Corporation produce a PowerPlex Y kit (23-locus) • To standardise population data specific sets of loci (often called minimum haplotypes) have been chosen by scientists in Europe and the US</li><li> Novel Y-STR loci are being continually described • One of interest is the multicopy locus DYS464 [1] » Occurs four times on the Y- chromosome in the highly palindromic region near the centre of the long arm » Highly advantageous for forensic casework where quantity of DNA is often limited » </li><li> There are well over 200 Y-STR loci that have been identified and are suitable for forensic purposes

 For most of the Y STRS there is only one copy of the sequence on the Y chromosome i.e. one allele per locus

 Unlike mtDNA the low mutation rate and the presence of only one copy of Y chromosome per cell, heteroplasmy is of no concern

 The term “hemizygous” may be used to denote the single- copy Y chromosome haplotype</li></ul>

Y<br><br><ul><li> useful for samples containing relatively small amounts of male DNA - e.g. sexual assaults. • Family studies – paternity, especially of long “distances” • Database searching for relatives.

95 </li></ul>