FRSC-3000: Lecture 1

RFLP with single-locus VNTR probes advantages:

• Excellent powers of discrimination

• Larger number of alleles at each locus which facilitates mixed-sample analysis

RFLP with single-locus VNTR probes limitations:

• Limited sensitivity

• Time-consuming (not automated)

• Not suitable with degraded DNA

• Requires grouping alleles into bins (statistical complications)

• Limited number of validated loci

What does RFLP detect?

A variable number of tandem repeats (VNTRs)

PCR-based methods advantages:

• Very small amounts of DNA template may be used

• Degraded DNA can be used as templates

• Large number of copies of specific DNA sequences can be amplified simultaneously with multiplex PCR

• Contaminant DNA will not amplify due to human-specific reaction setup

• Commercial kits available

PCR-based methods disadvantages:

• The target DNA template may not amplify due to PCR inhibitors

• Amplification may fail due to mutations in the binding region of the target sequence

• Contamination from other human DNA sources is possible

What is AFLP?

Amplified fragment length polymorphism

AFLP advantages:

• Improved sensitivity compared to RFLP because it uses PCR

• Many alleles which facilitates mixed sample analysis

• Discrete allele calling possible using allelic ladder (simplifies statistical analysis)

AFLP limitations:

• Large allele range with preferential amplification of small alleles

• Poor power of discrimination as a single locus

• Allele dropout in highly degraded DNA

• Gel separation and silver stain not automated (Time consuming)

Reverse Dot Blot Test advantages:

• Fast and simple when compared to RFLP

• Can analyze small or degraded DNA because it uses PCR

• No instrumentation needed after PCR

Reverse Dot Blot Test limitations:

• Poor power of discrimination with only six loci

• Mixture interpretation difficult

STR sequences account for ~ what percent of the total human genome?

3%

What are short tandem repeat markers (STRs)?

Accordion-like DNA sequences that occur between genes (they are non-coding regions of DNA)

Silver-Stained STRs advantages:

• Sensitive due to PCR

• Relatively rapid process

• Can use degraded DNA

• Lower start-up cost when compared to fluorescent STRs

Silver-Stained STRs limitations:

• Multiplex amplification and detection is limited to 3-4 loci because only one colour channel is available

• Both strands of DNA are detected leading to double bands which complicate interpretation

Fluorescent STRs advantages:

• Sensitive due to PCR

• Relatively rapid process

• Can use degraded DNA

• Multiplex PCR amplification and multicolour labelling

• Standardized sets of core loci

• Automated detection enables high-throughput processing

• The potential number of loci is very high

Fluorescent STRs limitations:

• Less discriminatory power per locus compared to VNTRs

• Increased possibility of contamination due to PCR

• Expensive equipment

• Stutter products and unbalanced peak heights

• Data interpretation complicated by artifacts

Between RFLP and PCR-based methods, which is faster?

PCR-based

Between RFLP and PCR-based methods, which requires less sample DNA?

PCR-based

Between RFLP and PCR-based methods, which requires dsDNA?

• Required for RFLP

• Can be used in PCR

Between RFLP and PCR-based methods, which can be automated with high-throughput processing?

PCR-based

How many cells are needed to recover a DNA profile?

The best results are with >100 cells, but they can be recovered from as little as a single cell.

What are the non-DNA-based forensic ID methods?

• Blood group testing

• Forensic protein profiling

What are the DNA-based forensic ID methods?

• RFLP length analysis of single and multi-locus markers

• PCR based sequence analysis via reverse dot blot

• PCR based length analysis via AFLPs, silver-stained STRs, and fluorescent STRs

• mtDNA sequencing

Blood group typing advantages:

• Rapid, simple tests

• Was the only test available for many years

Blood group testing limitation:

• Poor power of discrimination with few alleles (~1 in 10)

To be useful, DNA markers must be:

• Variable (polymorphic)

• Reproducible detection

What is the process of RFLP DNA testing?

• Cut DNA with restriction enzymes

• Separate fragments by size (length) via gel electrophoresis

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

• Strip the membrane and re-probe if needed