The Use Of Reduced Sized Pcr Products miniSTRs

In an article entitled 'Less is more - length reduction of STR amplicons using redesigned primers,' Wiegand and Kleiber (2001) demonstrated that highly degraded DNA as well as very low amounts of DNA could be more successfully typed using some new redesigned PCR primers that were close to the STR repeat compared to the established sequences that generated longer amplicons for the same loci. STR loci used in commercially available kits can extend past 400 bp in size (see Table 5.5). Most of this length however comes from flanking sequence surrounding the STR repeat of interest. PCR primers for larger sized STR markers have been moved away from the repeat region that imparts variability to the locus in order to fit into a desired size range for a particular multiplex assay (e.g., Krenke et al. 2002).

For example, the two PCR primers used for the PowerPlex 16 locus Penta D anneal 71 bp upstream and 247 bp downstream of the core AAAGA repeat. Amplification with these PCR primers generates amplicons in the size range of 376-449 bp with alleles ranging from 2.2-17 repeats (Krenke et al. 2002). When primers are brought to within 11 bp upstream and 19 bp downstream of the repeat region, the overall PCR product sizes drop by 282 bp to a range of 94-167bp for alleles 2.2-17 (Butler et al. 2003). Figure 7.2 illustrates this size reduction principle when creating reduced size STR amplicons or 'miniSTRs.' It is important to keep in mind that some loci can be reduced in size more than others (Table 7.1).

Several disadvantages do exist for miniSTRs. A major disadvantage is that only a few loci can be simultaneously amplified in a multiplex because the size aspect has been removed. Large multiplex assays like PowerPlex 16 pack four or more loci into a single dye color by shifting primers away from the repeat region to make larger PCR products. The 'miniplexes' created for amplifying miniSTRs have primers that are as close as possible to the repeat region and therefore typically only have one locus per dye color because all of the loci are about the same general size range of ~100bp (Butler et al. 2003).

Due to the fact that different PCR primers are in use with miniSTRs compared to conventional STR megaplexes, it is important that concordance studies be performed to verify that allele dropout from primer binding site mutations is rare or non-existent. This is performed by examining the geno-typing results to see if they are the same between the primer sets (see Chapter 6). Occasionally a point mutation or an insertion or deletion may occur in the flanking region outside of a miniSTR primer binding site which can lead to a

Conventional miniSTR

PCR primer fT^

miniSTR primer

Conventional PCR primer

Conventional STR test

1246.271 |254~37|

' 70 ' ' 75" ' s0' ' ' 85" "90" "95" "00 ' ' 105 ' "10 ' ' ii5 ' "20 ' ' 125 " i3o

I D16S539 I

6000 miniSTR assay (using 4000 Butler et al. 2003 primers)

Figure 7.2

(a) MiniSTRs or reduced sized amplicons for STR typing are created by designing PCR primers that anneal closer to the repeat region than conventional STR kit primers. (b) PCR product sizes, such as demonstrated here with D16S539, can be reduced by over 150 bp relative to conventional tests. MiniSTR assays can produce the same typing result as those from larger STR amplicons produced by kits often with greater success on degraded DNA samples.

Reference

Locus

miniSTR size

Size reduction

(alleles range)

over kit or

previous primers*

Hellmann et a/.

TH01

61-85 bp (alleles 5-10)

-103 bp

(2001)

TPOX

58-86 bp (alleles 6-13)

-157 bp

FES/FPS

81-105 bp (alleles 8-14)

-132 bp

Tsukada et a/.

TH01

74-98 bp (alleles 5-11)

-90 bp

(2002)

TPOX

107-135 bp (alleles 6-13)

-110 bp

CSF1PO

90-122 (alleles 7-15)

-194 bp

VWA

99-143 bp (alleles 10-21)

-53 bp

Butler et a/.

TH01

51-98 bp (alleles 3-14)

-105 bp

(2003)

TPOX

65-101 bp (alleles 5-14)

-148 bp

CSF1PO

89-129 bp (alleles 6-16)

-191 bp

VWA

88-148 bp (alleles 10-15)

-64 bp

FGA

125-281 bp (alleles 12.2-51.2)

-71 bp

D3S1358

72-120 bp (alleles 8-20)

-25 bp

D5S818

81-117 bp (alleles 7-16)

-53 bp

D7S820

136-176 bp (alleles 5-15)

-117 bp

D8S1179

86-134 bp (alleles 7-19)

-37 bp

D13S317

88-132 bp (alleles 5-16)

-105 bp

D16S539

81-121 bp (alleles 5-15)

-152 bp

D18S51

113-193 bp (alleles 7-27)

-151 bp

D21S11

153-211 bp (alleles 24-38.2)

-33 bp

Penta D

94-167 bp (alleles 2.2-17)

-282 bp

Penta E

80-175 bp (alleles 5-24)

-299 bp

D2S1338

90-142 bp (alleles 15-28)

-198 bp

Table 7.1 PCR product size reduction obtained with new primers in several miniSTR studies.

Table 7.1 PCR product size reduction obtained with new primers in several miniSTR studies.

■ 1111111111111111111111111111111111111111111111111111

225 230 235 240 245 250 255 260 265 270 275

*Comparisons between various studies were adjusted to be against the same 'kit or previous primers' (usually the AmpF/STR kits).

problematic (and undetectable) difference in a heterozygous allele call (see Butler et al. 2003, Drabek et al. 2004).

Regardless of these disadvantages, it is likely that miniSTRs will play a role in the future of degraded DNA analysis probably to help recover information that has been lost with larger loci from conventional megaplex amplification. With DNasel-digested DNA, miniSTR loci performed better than loci from a commercial STR kit (Chung et al. 2004). As will be described in Chapter 24, reduced size STR assays have helped make possible some of the World Trade Center victim identifications from burned and damaged bone samples (Schumm et al. 2004). Even telogen hair shafts, which contain very little nuclear DNA, have been successfully typed using reduced size STR amplicons (Hellman et al. 2001). New STR loci besides the CODIS markers and others that are currently used in forensic DNA typing are also being examined as potential miniSTR systems with a focus on loci possessing small alleles and narrow size ranges (Ohtaki et al. 2002, Coble and Butler 2005). Thus, a battery of additional assays should be available to aid researchers and forensic practitioners in the future when working with degraded DNA specimens.

PCR INHIBITION

Another important challenge to amplifying DNA samples from crime scenes is the fact that the PCR amplification process can be affected by inhibitors present in the samples themselves. Outdoor crimes may leave body fluids such as blood and semen on soil, sand, wood, or leaf litter that contain substances which may co-extract with the perpetrator's DNA and prevent PCR amplification. Textile dyes, leather, and wood from interior crime scenes may also contain DNA polymerase inhibitors.

Inhibitors can (1) interfere with the cell lysis necessary for DNA extraction, (2) interfere by nucleic acid degradation or capture, and (3) inhibit polymerase activity thus preventing enzymatic amplification of the target DNA (Wilson 1997). Occasionally substances such as textile dyes from clothing or hemoglobin from red blood cells can remain with the DNA throughout the sample preparation process and interfere with the polymerase to prevent successful PCR amplification (Akane et al. 1994, DeFanchis et al. 1988, Radstrom et al. 2004).

The result of amplifying a DNA sample containing an inhibitor such as hematin is a loss of the alleles from the larger sized STR loci or even complete failure of all loci. Some example inhibitors that interfere with PCR amplification are listed in Table 7.2. Samples containing PCR inhibitors often produce partial profile results that look similar to a degraded DNA sample (see Applied Biosystems 1998). Thus, failure to amplify the larger STR loci for a sample can be either due to degraded DNA where there are not enough intact copies of the DNA template or due to the presence of a sufficient level of PCR inhibitor that

Possible Forensic Source

PCR Inhibitor

Summary of some PCR

Blood

Heme (hematin)

Akane et al. (1994)

inhibitors. Some of these inhibitors are removed

Tissue and Hair

Melanin

Eckhart et al. (2000)

during routine DNA

Feces

Polysaccharides

Monteiro et al. (1997)

extraction methods, such as Chelex (see Chapter 3),

Feces

Bile salts

Lantz et al. (1997)

while others may need additional solutions

Soil

Humic compounds

Tsai and Olson (1992)

(see Radstrom et al. 2004). Most inhibitors

Urine

Urea

Mahony et al. (1998)

bind to and interfere with

Blue jeans

Textile dyes (denim)

Shutler et al. (1999)

polymerase activity.

reduces the activity of the polymerase. Reduced size STR amplicons can aid in recovery of information from a sample that is inhibited since smaller PCR products may be amplified more efficiently than larger ones.

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