Lecture 9: DNA degradation

Question 1

what stem DNA structure?

Answer 1

– A deoxyribose sugar.
– A phosphate group.
– A nitrogenous base

Question 2

what happens when tissue is showing advance stages of decomposition?

Answer 2

cellular structures have begun to break down, in doing so the cells release nucleases which degrade DNA.

Question 3

DNA is subject to nucleases from where?

Answer 3

– Endogenous nucleases released by host cells.

– Exogenous nucleases from microorganisms (bacteria and fungi) and environmental invertebrates.

Question 4

what 2 main processes breakdown DNA?

Answer 4

hydrolysis

oxidation

Question 5

what does hydrolysis first do?

Answer 5

removal of bases particularly purines , A and G

also bases with second amines, A, C and G, deamination leads to change of nucleotide

Question 6

what does oxidative damage lead too?

Answer 6

• lesions in the sugar-phosphate backbone
• chemical alterations of the bases
• Pyrimidines such as Thymine are more sensitive to oxidative damage than purines.

Question 7

Name 4 types of ancient DNA damage?

Answer 7

strand breaks
miscoding lesions vis hydrolysis +oxidation
cross links

Question 8

what’s the effect and solution if the strand of ancient DNA breaks?

Answer 8

low quantity survives, amplify is short 100-300bp

Question 9

whats the most important factor in preserving DNA?

Answer 9

Low temperatures

Question 10

what else plays a role in degradation rate ?

Answer 10

humidity
soil ph
chemicals

Question 11

how does humidity play a role in degradation?

Answer 11

water encourages growth of bacteria

Question 12

how does soil ph play a role in degradation?

Answer 12

Neutral and alkaline soils favour DNA preservation.

Question 13

how do chemicals ph play a role in degradation?

Answer 13

Specific chemicals prevent the action of degrading enzymes e.g. limestone in soil.

Question 14

what needs to be assessed when considering recovery of DNA from human remains?

Answer 14

state of degradation

Question 15

what happens if theres been severe degradation + long post mortem period ?

Answer 15

samples such as muscle, tissue and hair may be of limited use.

Question 16

what should be sued to find DNA if theres been severe degradation?

Answer 16

hard tissue such as bone, teeth (and nails) may be more successful at yielding a DNA profile

Question 17

what 2 things can been tissue be categorised as?

Answer 17

compact or spongy

Question 18

Most DNA in compact bone is located where?

Answer 18

osteocytes, these cells are the most common source of nucleated cells.

Question 19

Bone is useful as the ____ _____ helps to preserve DNA as well as the presence of ________ which stabilises DNA molecules.

Answer 19

physical barrier

hydroxyapatite

Question 20

Can DNA degradation occur in bone?

Answer 20

yes extraction can be complex

Question 21

why are teeth a good source of DNA?

Answer 21

well protected by enamel.

Question 22

what cells form teeth can be used?

Answer 22

Fibroblasts - pulp cavity, more sensitive to purefaction, humidity an bacteria.
Cementoblasts - cementum
Odontoblasts - in dentin mtdna

Question 23

how to assess degradation of hard tissue?

Answer 23

1. Morphological
    deformationandfragmentation 2. Structural
    loss of water and collagen 3. Chemical
    organicandinorganiccomponent 4. Biological
    AA racemisation

Question 24

How can degradation index be calculated?

Answer 24

using the ratio of the small and large autosomal target.

Question 25

whats the degradation index equation?

Answer 25

DI = conc of small DNA/conc of large DNA

Question 26

what’s an advantage of hard tissue?

Answer 26

it can be cleaned (sanded) to remove:
– any commingled remains
– Adhering inhibitors
– Bacterial contamination

Question 27

what’s the first stage of extracting from hard tissue?

Answer 27

Cleaning

• abrasion to remove outer surface
• washing with bleach and detergent
• expose to UV prevent amplification of exogenous DNA

Question 28

what’s the second and third stage of extracting from hard tissue?

Answer 28

2. Material is broken down into a powder by drilling or grinding under liquid nitrogen.
3. Powder is then decalcified using EDTA followed by cell lysis using proteinase allowing access to the enclosed DNA

Question 29

what’s the fourth stage of extracting from hard tissue?

Answer 29

Extraction using organic phenol-chloroform method or silica binding methods.

Question 30

whats Jörg Jenatsch case study ?

Answer 30

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.

Question 31

how wa the sample from Jenatsch case irradiated?

Answer 31

irradiated with UV to

decontaminate surfaces.

Question 32

what’s a challenge when it comes to bone and teeth?

Answer 32

ontain high amounts of PCR inhibitors

Question 33

why is contamination a major issue with ancient samples?

Answer 33

PCR will favour modern DNA over ancient DNA molecules.

Question 34

how is PCR efficiency will be reduced ?

Answer 34

number of DNA molecules break at a primer site or in between where the forward and reverse anneal

Question 35

Modifications to the DNA molecule can lead to what?

Answer 35

incorrect bases being inserted during extension by DNA polymerase.

Question 36

UV irradiation can cause what?

Answer 36

cross linking of thymine bases which will prevent the passage of DNA polymerase during the PCR.

Question 37

what’s one stochastic effect?

Answer 37

Preferential amplification–allelic drop out

Question 38

what’s another stochastic effect?

Answer 38

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.

Question 39

what’s a good alternative when no nuclear DNA ?

Answer 39

MitochondrialDNA

Question 40

what makes mtDNA useful?

Answer 40

highcopynumber

Question 41

what has a large impact on the success of the degraded DNA amplification success.

Answer 41

choice of DNA polymerase

Question 42

whats the most effective DNA polymerase for a degraded DNA sample?

Answer 42

Ex Taq HS, FastStart Taq, and PicoMaxx HF

Question 43

what can be redesigned to help amplify degraded DNA?

Answer 43

Primers

make flanking region closer to the STR repeat.

Question 44

why are SNPs not as useful to amplify degraded DNA?

Answer 44

bi-allelic nature means a larger number of SNPs are needed to give maximum discrimination power.

Question 45

what’s another way in profiling degraded DNA

Answer 45

target more protected areas of the genome.

Question 46

what stem bro protested area of DNA?

Answer 46

nucleosome core particle complex - packaged DNA

Question 47

How to avoid contamination?

Answer 47

• 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)