Molecular Markers

Question 1

Different types of DNA

Answer 1

MtDNA - animals and plants
CpDNA - animals
NDNA (nuclear) - chromosomes

MtDNA and cpDNA used for barcoding

Question 2

General overview of PCR process

Answer 2

1. Heat DNA 95C = dsDNA separates
2. Cool DNA 55C = primers bind to template
3. Polymerase enzyme adds complementary bases = identical copy of template formed
4. Each cycle doubles the number of copies
   30 cycles = 1mil copies

Question 3

Overview of mTDNA

Answer 3

• numerous in the cell (>300 per cell)
• circular
• haploid
• maternal clonal inheritance
• non-recombinant
• evolves 10x faster than genome

Question 4

Advantages of MtDNA as a molecular marker

Answer 4

• Easy to isolate and amplify

- good for evolutionary studies = different genes evolve at different rates

Question 5

Disadvantages of mtDNA as a molecular markers

Answer 5

• only provides info maternal dispersal/evolution
• copies also found in chromosomes = sequencing nDNA by mistake
• issues such as heteroplasmy are rare but can occur
  (MtDNA from sperm is included rather than just maternal in egg)

Question 6

Overview of nDNA as molecular marker

Answer 6

• large genome
• chromosomes
• bi-parental inheritance
• provides independent information for trees, population genetics and parentage…can have more than one marker per chromosomes = more statistical power
• SNPs
• copy number variants
• whole genome sequencing

Question 7

Different methods used to create molecular markers with nDNA

Answer 7

1. DNA fingerprinting
2. PCR based finger printing
   - AFLP (amplified fragment length polymorphism)
   - RAPD (random amplified fragment polymorphic DNA)
3. DNA profiling
   - microsatellites
   - SNPs
4. DNA sequencing
   - high throughput
   - massively parallel sequencing

Question 8

Advantages of nDNA as a molecular marker

Answer 8

• high variable bands in barcode
• lots of genomic locations can be assessed at the same time (loci)
• gives phylogenetic and population analysis more statistical power
• no prior knowledge of the genome is required

Question 9

Disadvantages of using nDNA as a modular marker

Answer 9

• cannot identify individual loci or type of inheritance

- unable to use non-invasive sampling..need high quality DNA

Question 10

2nd generation sequencing methods

Answer 10

Sanger sequencing
Illumina sequencing (bridge amplification)
454 sequencing
Shotgun sequencing

Question 11

3rd generation sequencing methods

Answer 11

PAC bio
Nanopore
Single molecule real time sequencing (SMRT)