Lecture 9: DNA degradation Flashcards
what stem DNA structure?
– A deoxyribose sugar.
– A phosphate group.
– A nitrogenous base
what happens when tissue is showing advance stages of decomposition?
cellular structures have begun to break down, in doing so the cells release nucleases which degrade DNA.
DNA is subject to nucleases from where?
– Endogenous nucleases released by host cells.
– Exogenous nucleases from microorganisms (bacteria and fungi) and environmental invertebrates.
what 2 main processes breakdown DNA?
hydrolysis
oxidation
what does hydrolysis first do?
removal of bases particularly purines , A and G
also bases with second amines, A, C and G, deamination leads to change of nucleotide
what does oxidative damage lead too?
- lesions in the sugar-phosphate backbone
- chemical alterations of the bases
- Pyrimidines such as Thymine are more sensitive to oxidative damage than purines.
Name 4 types of ancient DNA damage?
strand breaks
miscoding lesions vis hydrolysis +oxidation
cross links
what’s the effect and solution if the strand of ancient DNA breaks?
low quantity survives, amplify is short 100-300bp
whats the most important factor in preserving DNA?
Low temperatures
what else plays a role in degradation rate ?
humidity
soil ph
chemicals
how does humidity play a role in degradation?
water encourages growth of bacteria
how does soil ph play a role in degradation?
Neutral and alkaline soils favour DNA preservation.
how do chemicals ph play a role in degradation?
Specific chemicals prevent the action of degrading enzymes e.g. limestone in soil.
what needs to be assessed when considering recovery of DNA from human remains?
state of degradation
what happens if theres been severe degradation + long post mortem period ?
samples such as muscle, tissue and hair may be of limited use.
what should be sued to find DNA if theres been severe degradation?
hard tissue such as bone, teeth (and nails) may be more successful at yielding a DNA profile
what 2 things can been tissue be categorised as?
compact or spongy
Most DNA in compact bone is located where?
osteocytes, these cells are the most common source of nucleated cells.
Bone is useful as the ____ _____ helps to preserve DNA as well as the presence of ________ which stabilises DNA molecules.
physical barrier
hydroxyapatite
Can DNA degradation occur in bone?
yes extraction can be complex
why are teeth a good source of DNA?
well protected by enamel.
what cells form teeth can be used?
Fibroblasts - pulp cavity, more sensitive to purefaction, humidity an bacteria.
Cementoblasts - cementum
Odontoblasts - in dentin mtdna
how to assess degradation of hard tissue?
- Morphological
deformationandfragmentation 2. Structural
loss of water and collagen 3. Chemical
organicandinorganiccomponent 4. Biological
AA racemisation
How can degradation index be calculated?
using the ratio of the small and large autosomal target.
whats the degradation index equation?
DI = conc of small DNA/conc of large DNA
what’s an advantage of hard tissue?
it can be cleaned (sanded) to remove:
– any commingled remains
– Adhering inhibitors
– Bacterial contamination
what’s the first stage of extracting from hard tissue?
Cleaning
- abrasion to remove outer surface
- washing with bleach and detergent
- expose to UV prevent amplification of exogenous DNA
what’s the second and third stage of extracting from hard tissue?
- Material is broken down into a powder by drilling or grinding under liquid nitrogen.
- Powder is then decalcified using EDTA followed by cell lysis using proteinase allowing access to the enclosed DNA
what’s the fourth stage of extracting from hard tissue?
Extraction using organic phenol-chloroform method or silica binding methods.
whats Jörg Jenatsch case study ?
died 1639, body exhumed for second time in 2012, material from femur nd tooth, amplified using AmpF/STR Yfiller, Y23 for Y-STR analysis.
20 out of 23 Y matched male relatives. Y-SNP haplogroup’s of the skeleton and the male relatives haplogroups were the same.
how wa the sample from Jenatsch case irradiated?
irradiated with UV to
decontaminate surfaces.
what’s a challenge when it comes to bone and teeth?
ontain high amounts of PCR inhibitors
why is contamination a major issue with ancient samples?
PCR will favour modern DNA over ancient DNA molecules.
how is PCR efficiency will be reduced ?
number of DNA molecules break at a primer site or in between where the forward and reverse anneal
Modifications to the DNA molecule can lead to what?
incorrect bases being inserted during extension by DNA polymerase.
UV irradiation can cause what?
cross linking of thymine bases which will prevent the passage of DNA polymerase during the PCR.
what’s one stochastic effect?
Preferential amplification–allelic drop out
what’s another stochastic effect?
Jumping PCR
- DNA template does not contain the complete sequence due to degradation.
– Incomplete PCR product can act as a primer in later cycles leading to hybrid products.
what’s a good alternative when no nuclear DNA ?
MitochondrialDNA
what makes mtDNA useful?
highcopynumber
what has a large impact on the success of the degraded DNA amplification success.
choice of DNA polymerase
whats the most effective DNA polymerase for a degraded DNA sample?
Ex Taq HS, FastStart Taq, and PicoMaxx HF
what can be redesigned to help amplify degraded DNA?
Primers
make flanking region closer to the STR repeat.
why are SNPs not as useful to amplify degraded DNA?
bi-allelic nature means a larger number of SNPs are needed to give maximum discrimination power.
what’s another way in profiling degraded DNA
target more protected areas of the genome.
what stem bro protested area of DNA?
nucleosome core particle complex - packaged DNA
How to avoid contamination?
• Isolation of pre-PCR aDNA facility
• Cleaning of laboratory including UV- irradiation of
benches and equipment.
• Controls, repeat of negative PCR control.
• Repeat amplification of samples
• Comparisons to lab personnel (modern DNA)
