Lecture 7 Molecular Mapping and DNA Profiling.pdf

Molecular Mapping and DNA profiling Dr. Callum Vidor Acknowledgement of country Bundjil and Waang The Kulin Nation Note: this map of language groups is representative only and not recognised as official. https://www.timeout.com/melbourne/things-to-do/an-introduction-to-boon-wurrung-language-from...

Molecular Mapping and DNA profiling Dr. Callum Vidor Acknowledgement of country Bundjil and Waang The Kulin Nation Note: this map of language groups is representative only and not recognised as official. https://www.timeout.com/melbourne/things-to-do/an-introduction-to-boon-wurrung-language-from-aunty-fay-stewart-muir https://taungurung.com.au/creation-stories/ Micro- and Mini-satellite loci • There are many different short DNA sequences that repeat in tandem across the genome • Highly polymorphic • Loci have variant number of repeats between individuals • The chromosomes of an individual usually have variable repeats at the loci (heterozygotes) • Microsatellites aka Short Tandem Repeats (STRs) – repeats of 2-10 bp • Mini-satellites aka Variable Number of Tandem Repeats (VNTR) – repeats of 10 to 100 bp Micro- / Mini-satellite loci make for excellent molecular markers of • Highly variable allelic polymorphism degree • Easily detectable using gel electrophoresis via: t ease of detection 1. RFLP and probe based analysis (e.g. Southern hybridisation) - using the repeat sequence as a probe (VNTRs) 2. PCR amplification - using sequences on each side of the repeat as primers (STRs) high e.g. Indiv. 1 Indiv. 2 origin Arrows represent either (i) restriction sites if using Southern hybridization, or (ii) primer sites if using PCR origin 5 repeats 3 repeats + 2 repeats 3 repeats + Allele A1 - 2 repeats Allele A2 - 3 repeats Allele A3 - 5 repeats Indiv. 1: A1 A2 Indiv. 2: A2 A3 Note: satellite alleles are co-dominant – the heterozygote is recognizable Learning outcomes from this Lecture • Use genotyping and pedigree analysis to determine the phase of, and map distances between, molecular loci • Outline how molecular loci genotyping and mapping could be used in applications like genetic counseling or agricultural assisted breeding • Compare and contrast the techniques of VNTR DNA fingerprinting with STR DNA profiling, outlining their strengths and/or limitations • Provide applications of DNA profiling and how population genetics needs to be considered when interpreting DNA profiles Molecular Mapping and DNA profiling Mapping with molecular markers Dr. Callum Vidor How to calculate map distances using molecular markers • In Drosophila we analyse two DNA marker loci • Loci A with variable number of tandem repeats • Loci B with variable number of tandem repeats Genotype Parents: A1 A2; B1 B2 X A3 A3; B3 B3 Genotype Offspring: A1 A3; B1 B3 223 A1 A3; B2 B3 24 A2 A3; B2 B3 227 A2 A3; B1 B3 26 D t prop of individuals are the PARENTAL ARRANGEMENT Mapping of DNA markers using genotyping and pedigrees • Example: There are three linked DNA marker loci (A, B and C) each with several alleles. From the triple heterozygote I1, determine if the map distance between the three genes. (Note: The genotypes of I.2 and their gametes are not shown) A1 A2; B1 B2; C1 C2 I II 1 1 2 3 2 4 5 6 7 8 How do we determine the phase of the DNA marker alleles in the triple heterozygote (i.e. which chromosome has which allele – i.e. the parental arrangement)? Mapping of DNA markers using genotyping and pedigrees • If parental genotypes are know, parental combinations easier to identify A1 A3; B1 B3; C1 C3 A2 A4; B2 B4; C2 C4 1 A1 A2 B1 B2 C1 C2 I II 2 1 2 1 3 The genotype for closely linked genes or markers on a single chromosome or gamete is called its haplotype (haploid genotype). 2 4 5 6 7 8 Because I1 inherited A1, B1 and C1 from their father and not from their mother, they must be together on one chromosome. Similarly, A2 B2 and C2 must be on their other chromosome. Mapping of DNA markers using genotyping and pedigrees • Parental and recombinant gametes can then be determined by genotyping the offspring of I1 (Note: The genotypes of I.2 and their gametes are not shown) A1 A2 B1 B2 C1 C2 I II 1 2 A1 B1 C1 A2 B2 C2 1 2 3 A2 B1 C1 4 A1 B1 C1 5 A1 B1 C2 6 A1 B1 C1 7 A2 B2 C2 8 A2 B2 C1 1 recombination / 8 between A and B (in II.3), so map distance = 12.5 map units 2 recombinations / 8 between B and C (in II.5 and II.8), so map distance = 25 map units A B 12.5 C 25 Note – inaccurate if just use one pedigree! Mapping of DNA markers using genotyping and pedigrees • If parental genotypes are unknown - parental combinations can be assumed from offspring A1 A2 B1 B2 C1 C2 I II 1 A1 B1 C1 A&B (Genotype) A1 B1 A2 B2 A1 B2 A2 B1 Count 4 3 O 1 2 A2 B2 C2 1 2 3 A2 B1 C1 (Note: The genotypes of I.2 and their gametes are not shown) 4 A1 B1 C1 Proportion 8 0.875 I 0.125 5 A1 B1 C2 6 A1 B1 C1 7 A2 B2 C2 8 A2 B2 C1 Mapping of DNA markers using genotyping and pedigrees • If parental genotypes are unknown - parental combinations can be assumed from offspring A1 A2 B1 B2 C1 C2 I II 1 A1 B1 C1 A&B (Genotype) Count A1 B1 4 A2 B2 3 A1 B2 0 A2 B1 1 2 A2 B2 C2 1 2 3 A2 B1 C1 Proportion 7/8 = 0.875 1/8 = 0.125 (Note: The genotypes of I.2 and their gametes are not shown) 4 A1 B1 C1 5 A1 B1 C2 6 A1 B1 C1 7 A2 B2 C2 8 A2 B2 C1 Mapping of DNA markers using genotyping and pedigrees • If parental genotypes are unknown - parental combinations can be assumed from offspring A1 A2 B1 B2 C1 C2 I II 1 A1 B1 C1 A&B (Genotype) Count A1 B1 4 A2 B2 3 A1 B2 0 A2 B1 1 2 A2 B2 C2 1 2 3 A2 B1 C1 Proportion 7/8 = 0.875 1/8 = 0.125 (Note: The genotypes of I.2 and their gametes are not shown) 4 A1 B1 C1 5 A1 B1 C2 6 A1 B1 C1 7 A2 B2 C2 A&C (Genotype) Count A1 C1 3 A2 C2 2 A1 C2 1 A2 C1 2 8 A2 B2 C1 Proportion 5/8 = 0.625 3/8 = 0.375 Mapping of DNA markers using genotyping and pedigrees • If parental genotypes are unknown - parental combinations can be assumed from offspring A1 A2 B1 B2 C1 C2 I II 1 A1 B1 C1 A&B (Genotype) Count A1 B1 4 A2 B2 3 A1 B2 0 A2 B1 1 2 A2 B2 C2 1 2 3 A2 B1 C1 Proportion 7/8 = 0.875 1/8 = 0.125 (Note: The genotypes of I.2 and their gametes are not shown) 4 A1 B1 C1 5 A1 B1 C2 6 A1 B1 C1 7 A2 B2 C2 A&C (Genotype) Count A1 C1 3 A2 C2 2 A1 C2 1 A2 C1 2 8 A2 B2 C1 Proportion 5/8 = 0.625 3/8 = 0.375 B&C (Genotype) Count B1 C1 4 B2 C2 2 B1 C2 1 B2 C1 1 Proportion 6/8 = 0.75 2/8 = 0.25 Mapping of DNA markers using genotyping and pedigrees • If parental genotypes are unknown - parental combinations can be assumed from offspring A1 A2 B1 B2 C1 C2 I II 1 A1 B1 C1 A&B (Genotype) Count A1 B1 4 A2 B2 3 A1 B2 0 A2 B1 1 2 A2 B2 C2 1 2 3 A2 B1 C1 Proportion 7/8 = 0.875 1/8 = 0.125 (Note: The genotypes of I.2 and their gametes are not shown) 4 A1 B1 C1 5 A1 B1 C2 6 A1 B1 C1 7 A2 B2 C2 A&C (Genotype) Count A1 C1 3 A2 C2 2 A1 C2 1 A2 C1 2 8 A A2 B2 C1 Proportion 5/8 = 0.625 3/8 = 0.375 B C 12.5 B&C (Genotype) Count B1 C1 4 B2 C2 2 B1 C2 1 B2 C1 1 25 Proportion 6/8 = 0.75 2/8 = 0.25 Molecular Mapping and DNA profiling Applications of Molecular Mapping Dr. Callum Vidor Molecular mapping with DNA markers vs. genes DNA markers provide major advantages over genes for mapping: Number detected: Ease of scoring: Number of 'alleles’: Level of polymorphism: Genes DNA SNPs DNA Satellites Fewer Difficult Few Low Very many Moderate Up to 4 High Many Easy Many High Why use molecular loci to map chromosomes? • Traditional uses: • Provide ‘high resolution’ genetic maps of organisms • High resolution maps have >1 marker per map unit • Identify and clone genes using their map position • Determine if mutations affect different genes • Continuing uses: • Used to identify rare disease causing alleles • Allows tagging of desired alleles in plant/ animal breeding • Linkage with genetic markers can be considered in genetic counselling/ risk calculations • Assist in genome sequence assembly Klug et al., 12th Ed., Pearson Example: Marker Assisted Breeding • Nearby markers can be used as a ‘tag’ for a desirable allele/ trait e.g. there is an allele of the ‘Bold’ gene, b2, that is desirable in plants but is difficult to detect and only appears late in development • The Bold locus is in strong linkage with the A marker to one side, and the C marker to the other • form haplotypes A Bold A1 b2 C C4 • You genotype and select seedlings that only contain A1 and C4 • b2 should follow Example: Using markers to council on genetic risk e.g. Autosomal dominant condition – Huntington’s Disease (HD) • The gene involved in HD known to be closely linked to DNA marker ‘A’ I II III ? Example: Using markers to council on genetic risk e.g. Autosomal dominant condition – Huntington’s Disease (HD) • The gene involved in HD known to be closely linked to DNA marker ‘A” A1 A2 A3 A4 I A2 A3 A2 A4 A5 A6 II III A4 A5 ? Example: Using markers to council on genetic risk e.g. Autosomal dominant condition – Huntington’s Disease (HD) • The gene involved in HD known to be closely linked to DNA marker ‘A” I II h h A1 A2 h A2 III h A3 h H A3 h A2 A4 H h A4 A5 ? A4 A5 A4 allele is segregating with HD pathogenic variant h A6 Example: Using markers to council on genetic risk e.g. Autosomal dominant condition – Huntington’s Disease (HD) • The gene involved in HD known to be closely linked to DNA marker ‘A” I II h h A1 A2 h A2 III h A3 h H A3 h A2 A4 H h A4 A5 ? A4 A5 What is the chance that III1 has inherited the pathogenic HD allele? • Without genotypic information = 50% chance • Given they carry the A4 allele = 100% - chance of recombination • Assume A and HD gene are 20 mu apart • Chance with genotypic information = 100 – 20 = 80% chance h A6 Molecular Mapping and DNA profiling DNA profiling Dr. Callum Vidor The basis of DNA profiling • A number of situations require us to determine either the: • Identify of someone from the population or in comparison to another sample • Identify family relationships between individuals • Genomic DNA provides a fantastic tool in these situations because: • Genomic DNA is stable during life and the same DNA is found in all cells of the body • Excluding rare mutations • Each person’s genomic DNA is unique due to diversity of certain genomic regions • Related individuals share related DNA sequences via ancestry • Molecular methods are best suited to identify differences in genomic DNA between humans • Few differences can be observed at the phenotypic level O • Need to look in non-coding regions VNTR based ‘fingerprinting’ • The first major method of genome profiling analysis • Genomic DNA is processed to build a ’unique’ banding pattern based on mini-sattelite loci (VNTRs) a f to an unique individual • Produces a ‘fingerprint’ of bands • If higher number of loci used = increased probability pattern will be unique Brooker Genetics Analysis and Principles Fig 19.17 Hybridised with probe for sequence ‘V’ VNTR fingerprinting pros and cons • Pros O • Historically used to convict/exonerate individuals with regards to crime • Cons: • Require large amounts of DNA • DNA must be non-degraded • Can be hard to interpret with high certainty • Are similar bands really the same allele from the same locus? Repeat Type 1 Repeat Type 2 Alec Jeffreys CC BY 4.0 STR DNA profiling • Method still in use today • Use different and unlinked microsatellite loci (STRs) • small and highly variable in copy number (highly polymorphic) • PCR is used to amplify one locus at a time • Alleles defined unambigiously by the size of fragment produced ( based on number of repeats) • Produces a ‘genotype’ as each locus • Genotypes of multiple loci built into a unique ‘DNA profile’ Klug et al., Pearson Multiplexing in PCR based STR profiling • Many different PCRs in single reaction • Markers with difference in length separated by size • Markers with similar size identified based on different dyes incoporated into primers Colo • Automated detection using capilary based electrophoresis instead of bands p mostlikely Klug et al., 12th Ed Pearson zygous STR DNA profiling pros and cons • Pros • PCR amplification extremly sensitive, only small amounts of DNA needed • As small regions needed, works on highly degraded DNA • No ambiquity around alleles • Cons: alleles from of one those • Small amounts of contaminating DNA will easily amplify loci can be easily of another loci distinguish Alec Jeffreys CC BY 4.0 Armaron - CC BY-SA 3.0 Why not profile SNPs/SNVs? • SNPs are highly abundant across the genome • The lack of number of alleles at each loci (up to 4: A, T, G, C, commonly 2) mean many more loci need to be tested to build a unique profile • SNP locus is less Still has applications for highly degaded DNA, and in studies of lineage and evolution polymorphic than each individual STR Locus each individual Alec Jeffreys CC BY 4.0 DNA profiles are interpreted using probability • Probability based on population frequency of alleles is used to determine ’uniqueness’ of profile • Each locus is independant (unlinked) so probability is multiplied for each locus  The probability someone in the population has the exact DNA profile by chance is 0.00009%. * U.S. European-decent Database Klug et al., 12th Ed Pearson Molecular Mapping and DNA profiling Applications of DNA profiling Dr. Callum Vidor Content Warning • Contains reference to Forensic applications including: • Crimes of a sexual nature • Identification of human remains Alec Jeffreys CC BY 4.0 Forensic Applications – identifying/ruling out suspects • Commonly used to compare crime scenes/ crime samples with suspects Klug et al., 12th Ed Pearson Forensic Applications – identifying/ruling out suspects • Commonly used to compare crime scenes/ crime samples with suspects • Familial matching can be used • e.g. Convicted serial killer Lonnie Franklin aka ’The Grim Sleeper’ and his son’s DNA https://nebula.org/blog/gedmatch-genesis-review/ Forensic Applications – identifying/ruling out suspects • Commonly used to compare crime scenes/ crime samples with suspects • Familial matching can be used • e.g. Convicted serial killer Lonnie Franklin aka ’The Grim Sleeper’ and his son’s DNA • Personal genomics websites have been used to identiify suspects in crimes • Convicted serial killer Josseph DeAngelo aka ’The Golden State Killer’ and geneology database GED match https://nebula.org/blog/gedmatch-genesis-review/ Forensic Applications – identifying human remains • After crimes or large scale disasters, identification of human remains may be incredibly difficult by other methods • As only small amounts of DNA is required, the only viable method may be DNA profiling • People can be identified directly if an existing sample of cells or DNA profile is available • DNA profile can also be compared to family members of a missing person • e.g. A large portion of the victims of the Sep 11, 2001 World Trade Attacks were identified by DNA profiling alone, based on comparison to relatives or hair/blood samples Investigation of Relatedness or Paternity • Examples include: • Legal disputes over paternaty • Approval of immigration based on relatedness • Theft or unlawful use of agricultural breeding stocks • Confirming pedigree of pets or livestock • Tracking GMO crops • Determining source of poached wildlife • Analysing enviornmental populations • Inbreeding, Diversity Legal Considerations of DNA profiles – excluding vs proving • Exclusion of identity or relatedness is simple • If two DNA profiles are different, they cannot have come from the same person • DNA profiling has been instrumental in establishing innocence of those suspected or previously convicted of a crime • Complete proof of identity or relatedness is impossible • If two DNA profiles match, it can’t be concluded they came from the same person • Instead, a probability is provided that the DNA profile in question came about by chance • It’s used as just one line of evidence, among other pieces of evidence Legal Considerations of DNA profiles – errors • False inclusion of individuals • Relatives more likely to share alleles • Monozygotic twins – 100%, Parent/Child – 50%, Siblings: 40-60% (Average 50%) • Some alleles more common in certain populations / ethnic backgrounds • False exclusion of individuals • Technical problems with poor quality or low amounts of DNA • Contamination or a mixed source • Human error