Abstract

An optimized PCR-based system allowing the coamplification of the HLA DQα and the amelogenin genes has been developed and validated, enabling simultaneous identity testing and sex determination. Sensitivities below 100 pg of human DNA were obtained, using a convenient, high resolution agarose gel system and ethidium bromide staining. Comparison of several co-amplification methods revealed that the best sensitivities and most consistent results were obtained using a hotstart technique employing an inactivating antibody to Taq polymerase. HLA DQα typing results were reliably obtained using the co-amplification process. The sensitivity and ease of this system rendered it directly applicable to forensic analyses. The optimized techniques described here have been validated and successfully applied to forensic cases including People vs. Trujillo, in which the California Superior Court accepted these techniques as scientifically reliable and admissible. Work currently in progress has demonstrated that the described protocol may also be used to co-amplify the amelogenin gene with the AmpliType® PM (Polymarker) system, allowing identity testing at six loci in addition to sex determination.

References

1.
Nakahori
Y
,
Tanenaka
O
,
Nakagome
Y
.
A human X-Y homologous region encodes “amelogenin”
.
Genomics
 0888-7543
1991
;
9
:
264
-
9
.
2.
Bailey
D MD
,
Affara
N
,
Ferguson-Smith
M
.
The X-Y homologous gene amelogenin maps to the short arms of both the X and Y chromosomes and is highly conserved in primates
.
Genomics
 0888-7543
1992
;
14
:
203
-
5
.
3.
Nakahori
Y
,
Hamano
K
,
Iwaya
M
,
Nakagome
Y
.
Sex identification by polymerase chain reaction using X-Y homologous primer
.
Am J Med Genet
1991
;
39
:
472
-
3
.
4.
Sullivan
K M
,
Mannucci
A
,
Kimpton
C P
,
Gill
P
.
A rapid and quantitative DNA sex test: Fluorescence-based PCR analysis of the X-Y homologous gene amelogenin
.
Bio Techniques
1993
;
15
:
636
-
42
.
5.
Mannucci
A
,
Sullivan
K M
,
Ivanov
P L
,
Gill
P
.
Forensic application of a rapid and quantitative DNA sex test by amplification of the X-Y homologous gene amelogenin
.
Int J Legal Med
1994
;
106
:
190
-
3
.
6.
Casarino
L
,
De Stephano
F
,
Mannucci
A
,
Canale
M
.
HLA DQAI and amelogenin coamplification: A handy tool for identification
.
J Forensic Sci
1995 May;
40
(
3
):
456
-
8
.
7.
Perkin Elmer Corporation. AmpliType User Guide Version 2,
1993
.
8.
Buel
E
,
Wang
G
,
Schwartz
M
.
PCR amplification of animal DNA with human X-Y amelogenin primers used in gender determination
.
J Forensic Sci
1995 July;
40
(
4
):
641
-
4
.
9.
D'Aquilla
R T
,
Bechtel
L J
,
Videler
J A
,
Enron
J J
,
Gorczyca
P
,
Kaplan
J C
.
Maximizing sensitivity and specificity of PCR by preamplification heating
.
Nucleic Acids Res
1991
;
19
:3749.
10.
Chou
Q
,
Russell
M
,
Birch
D
,
Raymond
J
,
Bloch
W
.
Prevention of pre-PCR mispriming and primer dimerization improves low-copy-number amplifications
.
Nucleic Acids Res
1992
;
20
:
1717
-
23
.
11.
Kellogg
D E
,
Rybalkin
I
,
Chen
S
,
Mukhamedova
N
,
Vlasik
T
,
Siebert
P D
, et al
TaqStart™ antibody: “hotstart” PCR facilitated by a neutralizing monoclonal antibody directed against Taq DNA polymerase
.
BioTechniques
 0736-6205
1994
;
16
:
1134
-
7
.
12.
Sharkey
D J
,
Scalice
E R
,
Christy
K G
,
Atwood
S M
,
Daiss
J L
.
Antibodies as thermolabile switches: High temperature triggering for the polymerase chain reaction
.
Bio/Technology
 0733-222X
1994
;
12
:
506
-
9
.
This content is only available via PDF.
You do not currently have access to this content.