Forensic applications of RNA and methylation Flashcards
Name four different markers that can be used for identification
- STR
- Mini-STR
- Y STR
- mtDNA
What is the plex range of STRs, how many cycles are used in the PCR, how much DNA and from which samples is it commonly done?
- plex range: 15-24
- PCR cycles: 28-30
- DNA: 100pg
- samples: blood, hair roots, saliva with high quality DNA
What is the plex range of mini-STRs, how many cycles are used in the PCR, how much DNA and from which samples is it commonly done?
- plex: 9
- cycles: 30
- DNA: 50pg
- samples: degraded samples - bones
What is the plex range of Y-STRs, how many cycles are used in the PCR, how much DNA and from which samples is it commonly done?
- Plex: 17-23
- PCR: 30 cycles
- DNA: 100pg
- samples: sexual assault mixtures
What is the plex range of mtDNA, how much DNA and from which samples is it commonly done?
- Plex: HV1, HV2, HV3
- DNA: 1 pg, less than 1 cell, 100-1000 copies
- samples: hair shafts, bone samples with low copy number
Which areas are invovled in forensic analysis?
- genetics
- molecular biology
- proteomics
- genomics
- epigenetics
- population genetics
- biostatistics
Name two new applications in forensic genetics
- identification of type of biological fluid
- prediction of an unknown person’s age, physical appearance and ancestry
- to be used as investigative leads in a crime scene investigation
Which specific genes is used for circulatory blood recognition?
Glycoprotein A
is a major protein component of the human erythrocyte membrane
Which specific genes are used for menstrual blood recognition?
MMP11, MMP7
Zinc-dependent peptidases that take part in the disassembly of extracellular matrix and are expressed during tissue remodeling such as the female endometrium during the menstrual cycle
Which specific genes are used for semen analysis/the presence of spermatozoa?
- PRM1, PRM2 Protamine
- arginine rich proteins that help condensing the spermatid chromatin inside the nucleus
Which gene is used for seminal fluid recognition in the absence of spermatozoa?
TGM 4 Transglutaminase 4
Catalyses the permanent cross-linking of peptide bound glutamine residues. Specific expression to the prostate
Which genes are used for saliva recognition?
- HIS (histatin) and STATH (Statherin)
- Histatin: histidine-rich protein that is part of the immune defense of the oral cavity
- Statherin: stable acidic phosphoprotein of the saliva
- also: keratin 4 and 13; SPRR2A
Which genes are used for the regonition of vaginal secretions?
- HBD1 human beta defensin
- MUC4 Mucin 4
- defensin: antimicrobial protein produced by the neutrophils
- mucin: found in epithelial tissues of the body and belongs to the family of highly glycosylated proteins
Name some housekeeping genes that can be used to relate tissue specific markers to
- GAPDH
- ACTB
- SI5
- UCE
- TEF
- G6PDH
Which is tissue specific mRNA for skin?
Corneodesmosin
Loricin
Which are mRNA tests of blood (in general, I guess)
- Hemoglobin
- leucocyte markers
- CD93
- AMICA1
What can DNA methylation determine in forensics?
- differentiate monozygotic twins
- authentication of DNA samples
- parental origin of alleles
- identify tissue type/source
- cause and circumstances of death
- age of tissue donor
Name the two major epigenetic modifications
- histone modifications
- DNA methylation
- changes with increasing age
- contribute to age related disease
Name some methods for age estimation
- telomere length
- t-cell rearrangements
- RNA-biomarkers determine age based on four gamma globulin transcripts
- measure increase of a 4977 mtDNA deletion
- epigenetic changes
What is DNA methylation?
The addition of a methyl group 5’ position of the cytocine ring at a CpG site
Name some pro-aging factors (that induce methylation changes)
smoking, stress, alcohol
Name some anti-aging factors (that counteract methylation changes)
physical activity, antioxidant intake, calorie restriction diet
Pyrosequencing for methylation analysis - results at DNA basis
- unmethylated Cytosines appear as thymines (because C will be changed to Uracil)
- methylated cytosines appear as cytosines
- the ratio of C and T at a CpG site will give methylation level
Forensic age estimation tool
- quick and robust analysis
- Bisulfite conversion 30min
- PCR and pyrosequencing 3h
- high sensitivity
- 20ng down to 0.25 ng DNA
what has still to be done in the forensic age estimation tool?
- find an optimal set of markers - multiplex analysis
- accuracy in prediction, tissue variation
- sensitivity and degraded samples
How can you fake DNA?
- PCR - separate amplification of 10 loci
- WGA - multiple displacement amplification
- cloning
- amplified individual alleles and cloned them
- plasmids were purified and genotyped
- tubes with separate alleles in trillions of copies
- allele library for all 16 loci in the kit
- create any profile
How can you introduce DNA into blood and saliva?
- red blood cells separeated from white by centrifugation
- mixed with artificial DNA
- dripped and dried
- saliva extract without cells (the top phase after centrifugation)
- mixed with artifical DNA
- applied to ski-mask etc
What does the DNA autentication assay contain?
- bisulfite treatment
- sanger sequencing
- evaluation
- in vivo material: methylated and unmethylated DNA
- in vitro material: only unmethylated DNA
