Biohistory Flashcards
Which techniques have a slow speed of analyis and a high power of discrimination?
- RFLP mutli locus probes
- RFLP single locus probes
Solutions for mixtures
- Y-chromosome STRs
- Y-chromosome SNPs
NGS promises the simultaneous analysis of:
- whole exomes
- complete genomes
- whole transcriptomes
- whole methylomes
- many selected targets
Handling inhibition in bones
- removal during extraction
- silica or BTA purification is efficient
- additional clean up by Microcon etc if needed
- counteract in PCR
- addition of BSA
- extra enzyme
- choosing a different enzyme
- dilution of samples
- inhibitors can be monitored by real-time PCR
What is the most common mtDNA haplogroup in Europeans?
H (40-50%)
Decoding procedure
- e.g. cash in transit case
- sequence send to UK
- decoded in UK
- code connected to batch in belgium
- Belgium provides information about who got the cash
What is the use of forensic DNA testing in forensic cases?
- analyse evidence samples at a crime scene
- exonerate innocently accused or convicted
- match traces with DNA profiles in database with convicted/suspects
How can DNA damage be repaired?
With UNG treatment
UNG: Uracil N-glycosylase
At high degree of damage, positions with C/T mixtures seen in mtDNA sequence analysis
- C–> T changes via U, introduced by deamination
- UNG treatment can reduce the number of damaged molecules reducing the C/T mixtures
- UNG removes deaminated C-residues
How is mtDNA assigned into haplogroups?
Defined by certain SNPS in the hypervariabe and coding region
Name reasons why analysis of skeletal remains may be very challenging
- low template amounts
- highly degraded DNA
- damaged DNA
- contaminated
- precious samples - limited availability
–> optimal protocol for all steps is thus highly important
Strategies to overcome inhibition
- counteract inhibitors
- dilution of the DNA extract
- addition of BSA or betaine
- addition of extra enzyme
- use of an inhibitor resistant enzyme
- performing a more efficient removal through the extraction procedure
- making sure that EDA is removed
- inhibitors are smaller molecules than DNA –> filter
- inhibitors may bind to dsDNA –> denature by NaOH treatment
Which techniques are fast in analysis, but have a low power of discrimination?
- poly marker
- D1S80 single STR
- DQalpha
- ABO blood groups
Can solvents, ageing, bleaching or UV radiation wash-out DNA tags ?
no
Which techniques have a slow speed of analysis and a low power of discrimination?
- mtDNA
Name some causes for DNA damage and degradation
Generally occurs with time, but may be facilitated by:
- high humidity
- high temperature
- UV-radiation
- chemicals
- microorganisms
What affects DNA decay?
- release of cellular enzymes
- bacteria
- free radicals
How many SNPs are predicted in the HIrisPlex and from how many genes? What does it predict with high accuracy?
- 24 SNPs from 13 genes
- red and black hair
- blue and brown eyes
Which techniques have a fast speed of analyis and a high power of discrimination?
- SNPs
- mini STRs
- multiplex STRs (highest power)
How does the DNA tagging system work?
One example (there are different systems)
- unique signature between primer regions
- billions of available codes - 20nt unique region wtih 4 possible variants in each position
- totally invisible solution
- tags are unknown to the user
- the sequence of tags are easily read by pyrosequencing analysis
Which markers can be analysed with NGS?
- STRs
- SNPs
- X- and Y-chromosome markers
- the mt genome
at the same time in one shot!!
Name some famous persons where DNA analysis was used
- Josef Mengele
- The Romanovs and the missing children
- Jesse James
- The Evangelist Luke
- Carin Göring
- Kopernikus
Name causes of PRC inhibition
- co-purified compounds in bones or from soil
- humic compounds
- chemically altered carbohydrates
- heavy metals (iron, copper, cadmium and lead)
- collagen and chondroitin
- many other compounds/cheicals for other types of samples
- heme, polysaccharides, proteinases, melanin
NGS analysis is suitable for
- high quality samples
- small amounts/degraded samples
- mixed samples
Security marking with DNA - give examples of what can be DNA tagged
- security ink for bank notes
- label lega documents
- label oil in order to detect spill
- brand protection, e.g. clothes, perfume
- poperty marking, e.g. jewelry, valuables
- museum collection protection
- personal protection
Describe the DNA analysis process of DNA labeled security ink
- Cut banknote to extract it in water
- simple extraction, washing
- PCR
- binding of ssDNA to magnetic beads
- pyrosequencing with cyclic dispensation
Give three examples for DNA damage/degradation
- double strand breaks
- oxidative damage
- hydrolytic damage
Dilution may reduce the inhibitor concentration and increase DNA yield
Addition of BSA may increase DNA yield
Solutions for degraded DNA and small amounts of DNA
- miniSTRs
- SNPs - shorter fragments
- mtDNA - more copies
- real-time quantification
Which are the most susceptible bases for breakage of glycosidic base sugar bonds that lead to base loss and single stranded nicks at the basic site?
Guanine, followed by thymine
