Sso Probes And Linear Array Typing Assays

Table 10.5 (facing)

Methods for screening mtDNA variation (see Butler and Levin 1998 and Budowle et al. 2004).

One of the most widely used screening assays for assessing mtDNA variation used to date are the sequence-specific oligonucleotide (SSO) probes originally designed by Mark Stoneking and colleagues in 1991. Rather than sequencing the entire HV1 and HV2 regions, the most polymorphic sites are examined through hybridization of PCR products to oligonucleotide probes designed to anneal to different variants. The original paper describes 23 probes across nine regions that permit evaluation of variation at 14 different nucleotide positions (Stoneking et al. 1991). The sites that are probed include 16126, 16129, 16217, 16223, 16304, 16311, 16362, 73, 146, 152, 195, 199, 247, and 309.1. A number

Technique

Description

Reference

Sequence-specific oligonucleotide (SSO) dot blot assay

Mini-sequencing

Single-strand conformational polymorphism (SSCP)

Low-stringency single-specific-primer PCR (LSSP-PCR)

PCR-restriction fragment length polymorphism (PCR-RFLP)

23 SSO probes testing 14 sites within nine regions from HV1 and HV2; 274 mtDNA types observed among 525 individuals from five ethnic groups

Single base primer extension with fluorescent ddNTPs and poly(T)-tailed primers to yield different electrophoretic mobilities; 10 substitution and two length polymorphisms measured in the control region; 65 haplotypes observed from 152 British Caucasian samples

Differences in DNA secondary structure are detected on a native polyacrylamide gel; 25 mtDNA types observed among 45 Spanish individuals tested

Following regular PCR, a single primer and a low annealing temperature are used to generate a 'signature' pattern; for 30 unrelated individuals, all signature patterns were different across the control region (1024 bp)

A 199 bp region of HV1 is digested with Rsal; 19 unrelated mother-child pairs were examined with an 8% probability of a random match

Alonso et al. (1996)

Barreto et al. (1996)

Denaturing gradient gel electrophoresis (DGGE)

Affymetrix high-density DNA chip hybridization array

Pyrosequencing

SNaPshot (minisequencing)

Denaturing HPLC

Luminex 100 liquid bead array

Two DNA samples are mixed and run on a denaturing gradient gel; heteroduplexes, which travel more slowly through the gel, may be separated from the homoduplexes; samples that differ at a single location have been resolved

135 000 probes complementary to the entire mtGenome are contained on a microchip for parallel processing through hybridization

Sequencing by synthesis over ~50 nucleotides per reaction through an enzyme cascade that produces visible light; a total of 4 HV1, 4 HV2, and 11 coding region reactions were run

Allele-specific primer extension with 11 coding region SNPs combined into a single multiplex amplification and detection assay

HV1 and HV2 PCR products for a known and an unknown sample source are generated and then mixed together; samples that differ from one another by at least one nucleotide will form a heteroduplex on the HPLC

30 SNPs within HV1 and HV2 are examined by allele-specific hybridization with SSO probes attached to different colored beads that are separated using flow cytometry

Andreasson et al. (2002)

Vallone et al. (2004)

LaBerge et al. (2003)

www.marligen.com

LINEAR ARRAYs

Reverse dot blot hybridization with lines instead of dots using 18 SNPs in the same general probe regions as Stoneking et al. (1991)

Gabriel et al. (2003)

Figure 10.10

(a) Results schematically displayed of a known (K) reference and a question (Q) sample that do not match one another using the Roche LINEAR ARRAY mtDNA HVI/ HVII Region-Sequence Typing Strips. (b) Types are reported as a string of numbers representing the LINEAR ARRAY probe results. Failure of the PCR product to bind to a probe region (e.g., HVIE in sample Q) is referred to as a 'blank', is reported as a zero in the string of numbers, and is due to polymorphisms in the sample near the probe site that disrupt hybridization. Weak signals such as indicated by the arrow for 189 in sample Q are also due to a closely spaced polymorphism that disrupts full hybridization of the PCR product to the sequence-specific probe present on the LINEAR ARRAY.

of population studies have been conducted with these SSO probes including an examination of 2282 individuals from North America (Melton et al. 2001).

The original SSO probe assay required that the PCR products be attached through UV cross-linking to a nylon membrane and then each radioactively labeled probe was individually hybridized at different temperatures and finally exposed to autoradiographic film for several hours (Stoneking et al. 1991). Roche Molecular Systems (Alameda, CA) has converted the SSO probe assay into a more workable format involving colorimetric detection (e.g., Gabriel et al. 2001b). In a 'reverse dot blot' format, the SSO probes are attached to the nylon membrane in a linear array of spatially resolved lines of probes. Biotin-labeled PCR products are washed over nylon membrane strips containing immobilized SSO probes in the linear array and hybridized under uniform conditions. A streptavidin-horseradish peroxidase enzyme conjugate coupled with 3,3',5,5'-tetramethyl-benzidine creates a light blue colored precipitate using the same chemistry described for HLA-DQa reverse dot blot SSO probes (Saiki et al. 1989).

Figure 10.10 illustrates the probe layout for the LINEAR ARRAY Mitochondrial DNA HVI/HVII Region-Sequence Typing Kit now available from Roche Applied Sciences (Indianapolis, IN). The final linear array format examines 18 SNPs with 33 SSO probes present on 31 different lines. The Roche SSO probe sites are shown in Figure 10.6.

Two hypothetical results are illustrated in Figure 10.10 for non-matching K and Q samples. The K sample reported type of 1-1-1-1-1-1-1-1-1-1 is equivalent to the Cambridge Reference Sequence (see Figure 10.6). The Q sample possesses

HVI HVII I-II-1

16093 IA IC ID IE IIA IIB IIC IID 189

123 12 12 1245 12

Mil

1 1 2 3 4 5 6 7 1 1 1 1 1 1 1 1 1 1 1 1

1 2 1 1

K

'blank'

(b) Reported Types

a different pattern and therefore can be excluded from the K sample. Notice that probe IE within HVI did not produce a signal from any of the three possible probes. This result is referred to as a 'blank' and occurs due to additional polymorphisms that are present in close proximity to the polymorphic sites designed for detection in the assay. These additional polymorphisms disrupt hybridization of the PCR product and therefore no signal is seen for any of the probes in HVIE. Likewise weak (w) signals such as the 'w1' type are caused by mismatches between the PCR products and the SSO probes attached on the nylon strip.

Results from screening assays, such as the LINEAR ARRAY system described above, can be considered presumptive tests. They are useful in eliminating samples that can be excluded from one another. However, full HV1/HV2 sequencing would normally be performed to confirm any matches and see if differences outside of the SSO probe regions exist.

POPULATION DATABASES

Population databases play an important role in estimating the expected frequency of mtDNA haplotypes that are observed in casework when a suspect's mtDNA sequence matches that of an evidentiary sample. A great deal of effort has been expended to gather information from thousands of maternally unrelated individuals in various population groups around the world. Having high quality information in the database is also important in order to make a reliable estimate of the frequency for a random match.

Was this article helpful?

0 0
Stammering Its Cause and Its Cure

Stammering Its Cause and Its Cure

This book discusses the futility of curing stammering by common means. It traces various attempts at curing stammering in the past and how wasteful these attempt were, until he discovered a simple program to cure it. The book presents the life of Benjamin Nathaniel Bogue and his struggles with the handicap. Bogue devotes a great deal of text to explain the handicap of stammering, its effects on the body and psychology of the sufferer, and its cure.

Get My Free Ebook


Post a comment