PCR of challenging samples

Question 1

challenging samples

Answer 1

• DNA will not always be of optimum quality
• Old samples
• Environmental conditions
• Other DNA/ substances contaminating sample
• Can all affect amplification by PCR

Question 2

DNA degradation

Answer 2

• During decomposition, DNA templates can become fragmented or chemically modified
• This reduces the number of intact target fragments available for PCR
• May lead to PCR failure or poor amplification

Question 3

enzymatic degradation

Answer 3

• Enzymes internal and external to cell can breakdown DNA
• Nucleases
• Some associated with cell death (internal)
• Some in the environment (external)- microorganisms

Question 4

non enzymatic degradation

Answer 4

• Hydrolysis- breaking of a bond using water. In DNA the base-sugar bond is most susceptible to hydrolytic attack leading in the loss of a base
• Oxidative reactions- oxygen-derived free radicals can modify bases, blocking PCR
• Radiation can cause lesions to DNA molecule

Question 5

sample storage

Answer 5

• Once sample has been taken, the storage conditions can be controlled
• Keep sample dry-reduce bacterial growth and limit hydrolytic enzyme activity
• Constant low temperature will increase DNA longevity

Question 6

FTA Cards

Answer 6

• Room temperature storage
• Apply sample to FTA matrix and nucleic acids are captured and stabilised
• Successful amplification of blood after 22 years on FTA card

Question 7

3 steps to pure nucleic acids

Answer 7

spot, punch and purify

Question 8

exposure to high temperatures

Answer 8

• High temperatures will cause the covalent bonds within the DNA strands to break
• A study has found that this begins at 130◦C and that complete degradation occurs at 190◦C (Karni et al 2013)

Question 9

What can you do about degraded DNA

Answer 9

• Not much
• It is important to preserve samples as well as possible
• MiniSTRs have been developed which may have more amplification success with degraded DNA

Question 10

MiniSTRs

Answer 10

• Possible to reduce size of amplicon by putting primers closer to STR- advantage because of degraded DNA

Question 11

Low copy number (LCN)

Answer 11

• High sensitivity DNA profiling
• Increase the number of cycles >34
• Possible to obtain a profile from less than 100pg DNA (maybe as little as one cell)
• Extreme care required
• Samples very susceptible to contamination
• Interpretation of profiles much more complicated
• Caddy report- important to forensic scientists

Question 12

PCR inhibitors

Answer 12

• Humic compounds in soil, water and sediments
• Tannic acid in leather
• Haem in blood
• Collagen in bone
• Excessive DNA
• Heavy metals

Question 13

PCR inhibitions

Answer 13

• Inhibitors can hinder amplification in different ways
• By binding to and inactivating template
• Affect taq enzyme activity
• Applied biosystems quantifiler real time
  PCR quantification method has an internal PCR Control (IPC) that identifies samples that may contain inhibitors

Question 14

sample purification

Answer 14

• Try to remove inhibitors at the extraction stage
• Various approaches using magnetic beads, filters, gel
• Some substances will co-purify

Question 15

sample dilution

Answer 15

• By diluting the sample you can decrease the concentration of the inhibitor
• However, you will of course dilute the DNA as well!