Chapter 1-3: Overview and History of DNA Typing

What are the possible outcomes of evidence exmination?

• Exclusion (no match)

• Failure to exclude (“match” or “inclusion”)

• Inconclusive result

What does exclusion mean in terms of DNA testing?

Genotype comparison shows profile differences that can only be explained by the two samples originating from different sources

What does failure to exclude mean in terms of DNA testing?

Statistical evaluation of the significance of the match is usually reported with the match report

What does inconclusive mean in terms of DNA testing?

Might be reported if two analysts remain in disagreement after review and discussion of the data and it is felt that insufficient information exists to support any conclusion

What is a locus?

A specific region of DNA

What is a DNA profile?

The genotypes obtained at multiple loci

What makes up DNA?

• Sugar backbone

• Phosphate groups

• 4 nucleotide bases

How is the double helix of DNA formed?

Two DNA strands form hydrogen bonds

What creates the formation of adjacent nucleotides?

Phosphodiester bonds

What are the bonds between the pairing nucleotides?

• A-T has 2 bonds

• G-C has 3 bonds

What is a restriction enzyme?

An enzyme that recognizes a specific sequence of DNA (4-8 bp) and can detect sequence variation by the presence/ absence of a restriction cut

What do restriction enzymes produce?

A double-stranded cut (sticky or blunt)

What is the human genome?

• 23 pairs of chromosomes + mtDNA

• 22 nuclear DNA

• 2 sex chromosomes (X, Y)

What are the types of DNA polymorphisms?

• Sequence polymorphism

• Length polymorphism (bp repeats)

What are DNA markers?

Detectible variants of DNA

What must DNA markers do to be useful?

• Variable (polymorphic)

• Reproducible detection

How many core STR markers used in forensics?

20m+ sex typing

How do you calculate the total number of possible genotypes based on the number of alleles per locus/loci?

• Homozygous genotype = n alleles

• Heterozygous genotype = n(n-1)/2

• n + n(n-1)/2 = possible genotypes (for a marker with n alleles per locus)

How do you calculate the mass of DNA found in a cell?

• MM of DNA bp = 618g/mol

• 3.2 × 10⁹ bp in a haploid cell

• 152 diploid cells in 1ng of human DNA

1. (bp in haploid cell) x (MM of DNA) = 1.98 × 10¹²g/mol

2. (1.98 × 10¹²g/mol) x (avogadro’s constant) = grams of genomic DNA → pg

3. (1000 pg of genomic DNA)/ pg of genomic DNA = # of copies of each locus

4. # of copies of each locus/152 = # per 152 diploid genomes

What are the two main types of DNA markers?

• Non-DNA-based

• DNA-based

What technology does non-DNA-based include?

• Blood group testing

• Forensic protein profiling

What technology does DNA-based include?

• RFLP

• PCR-based (sequence and length)

• Mitochondrial DNA sequencing

What typing techniques have low power of discrimination?

• mtDNA

• DQα

• single STR

• ABO blood groups

What are the advantages of ABO blood group typing?

• Simple and rapid tests

• Only test available for many years

What are the disadvantages of ABO blood group typing?

• Poor power of discrimination with such few alleles.

What does RFLP-based DNA and Southern Blot detect?

Variable number tandem repeat (VNTRs)

What is the process for RFLP-based DNA and Southern Blots?

• Cut DNA with restriction enzymes

• Separate fragments differing in length by gel electrophoresis

• Detect length-based differences in fragments of interest with a radioactive probe

• Strip membrane and re-probe as necessary

What are the advantages of RFLP with single-locus VNTR problems?

• High powers of discrimination

• Large numbers of alleles at each locus, facilitating mixed-sample analysis

What are the disadvantages of RFLP with single-locus VNTR problems?

• Limited sensitivity

• time-consuming process that cannot be automated

• Not suitable with degraded DNA samples due to the high molecular weight needed

• Essentially continuous allele sizes - statistical complications and difficulty of interpretation

• Limited number of validated loci - limited value in distinguishing siblings

What are the advantages of PCR-based methods?

• Very small amounts of DNA template may be used

• DNA degraded to fragments can serve as an effective template for amplification

• Contaminant DNA will not amplify due to human-specific primers

• Commercial kits are available

What are the disadvantages of PCR-based methods?

• DNA template may not amplify due to PCR inhibitors in the extracted DNA

• Amplification may fail due to sequence mutations in the primer-binding region

• Contamination

What is AFLP?

• Amplified fragment length polymorphism

• PCR products separated on a polyacrylamide gel and detected with silver staining

What are the advantages of AFLP?

• Improved sensitivity compared to RFLP because it uses PCR

• Facilitates mixed-sample analysis

• Discrete allele calling possible - simplifies statistical interpretation

What are the disadvantages of AFLP?

• Large allele range, thus challenging to multiplex and have preferential amplification of smaller alleles

• Poor power of discrimination as a single locus

• Allele dropout

• Gel separation and silver-stain detection are not able to be automated or high-throughput sample processing

What are DQA1 dot blot tests?

Allele-specific probes that find sequence polymorphisms

What are the advantages of reverse dot blot tests?

• Fast and simple

• Capable of analyzing small or degraded samples because it used PCR

• No instrumentation needed after PCR

What are the disadvantages of reverse dot blot tests?

• Poor power of discrimination due to only 6 loci

• Mixture interpretation is difficult

What are STR Markers?

• Short tandem repeat markers

• An accordion-like DNA sequence that occurs between genes

What are the advantages of silver-stained STRs?

• Sensitive due to PCR

• Relatively rapid process

• Works well with degraded DNA

• A lower start-up cost

What are the disadvantages of silver-stained STRs?

• Multiplex amplification and detection are limited to 3 to 4 loci

• Both strands of DNA are detected, leading to double bands with some loci that can complicate the interpretation

What are fluorescent STRs?

Fluorescently labeled PCR primers to amplify and detect short tandem repeat (STR) regions in DNA

What are the advantages of fluorescent STRs?

• Sensitive

• Rapid process

• Works well with degraded samples

• Multiplex PCR amplification allows high power of discrimination

• Standardized sets of core loci

• Automated detection

• Large number of loci

What are the disadvantages of fluorescent STRs?

• Less discrimination power compared to VNTRs

• Possibility of contamination from stray DNA due to PCR process

• Expensive equipment

• Stutter products and unbalanced peak heights

• Data interpretation must account for artifacts