application of the ipcrp method for genotyping of male dna
TRANSCRIPT
Application of the IPCRp Method for
Genotyping of Male DNA Obtained by
Pressure Cycling Differential Extraction
Pero Dimsoski, Vanessa Martinez,
Deepthi Nori, and Bruce McCord
Florida International University
Miami, FL
Introduction
• NIJ is currently spending millions on DNA backlog
reduction and recovery of degraded evidence.There are some 20,000 untested rape kits sitting on evidence shelves in police department across Texas…
The New York Times January 3, 2013.
• A big issue is difficult extraction methods relying on
specific buffers, sonification and other steps to isolate
and remove the DNA from substrates.
• Efficiency and specificity of extraction are an important
issue especially with sexual assault and touch samples.
• We are exploring pressure based extraction coupled with
improvements of amplification and detection methods.
Alternatives to differential
extraction• Y STR typing.
• Laser microdissection.
• Cell sorting via Microdevices
or flow cytometry.
• Pressure cycling.
Cycles of pressure pulses are used to disrupt cell membranes and combined with specific buffers to isolate individual cell components.
a) Pressures can range from 5-45K PSI with up to 100 cycles.
b) Temperature and buffer conditions are varied to optimize release and digestion of cells.
a) Samples are placed in special “pulse “ tubes which permit application to individual samples.
What is pressurized
extraction?
Pulse Tubes
Fixed cap
Pulse tube
Ram
• Moveable Ram transfers pressure
to sample ( )+
_
Cycles of pressure and release are applied by a ram to a pulse tube.
Duration, # of pulses can be controlled.
• Sperm and epithelial cells, based on their different composition, should respond differently to pressure cycling.
– Epithelial cells are larger, with more diffuse structures. They should be more distorted by pressure, and thus more sensitive to its effects.
– Sperm DNA is associated with protamines, proteins with a high cysteine content, crosslinked with disulfide bridges– dense packing of DNA (12-18% cysteine).
– Epithelial cell nuclei are surrounded by histone proteins. These are not as cross linked as protamines – less dense packing
(0.2% cysteine).
Our hypothesis:
Sperm cell
Buccal Epithelial cell
Experimental design
SAMPLES
Sperm cells and Epithelial cells suspended in 1X PBS
buffer (pH 7.4)
CELL LYSIS
Pressure cycling using Barocycler® NEP 2320
vs Standard Proteinase K digestion
PURIFICATION
Phenol-Chloroform-Isoamyl alcohol extraction
or DNA IQ
QUANTITATION
Real-time PCR analysis using Plexor® HY system (Promega Corporation)
Key issue: controlling cell quantities and recovery of DNA
Use of real time PCR and extraction controls to provide confidence in the results.
Initial PCT- Microscopic studies
Cells stained with 0.4% Trypan blue (dye exclusion method) following
Pressure treatment
Color indicates PCT treatment is causing take-up of dye
Vaginal epithelial cells Sperm cells
Cell Visualization in PBS
Effect of temperature and added reagents
N=3, error bar= 1 SD
Marked improvement in recovery and interesting selectivity with DTT, temp, sarkosyl
Alternate reducing agent - TCEP
• Advantages to TCEP
• Water solubility
• Odorless
• Wide pH range
• Resistant to air oxidation
• Disadvantage:
TCEP is not particularly
stable in phosphate
buffers prepare fresh
daily.Mechanism: Bhasin, J Biological Chem., 279 (44)
5865–45874, 2004.
Goal: improve reduction of dithol linkages for sperm protamines
A comparison: DTT vs. TCEP
Dithiothreitol (DTT)Tris (2-carboxyethyl)phosphine (TCEP)
Switching to TCEP caused an increase in selectivity between sperm cell and epithelial cell lysis
Recovery of mixed DNA (5/1) Epithelial cells/ sperm
cells from the swab alkaline extraction
Male control
Female control
Following PCI extraction
Pressure cycling aided extraction can increase the male fraction in mixtures.
How to obtain a “cleaner” male genotype?
Enrichment of male fraction by immunomagnetic
capturing of female fraction
• Control sample created by adding sperm cells in 1x PBS.
• Epithelial cell specific antibodies added.
• Magnetic separation.
• Supernatant and captured cells collected.
• Pressure cycling for further enrichment and purification.
• PCIA extraction.
• Quantification (Plexor) and Analysis by PowerPlex16.
How to increase male fraction?
Immunomagnetic capturing of female fraction
• Antibodies attached to specific cell receptors
• Magnetic particles attach to the antibody complex
• The sample is inserted into a magnet
• Specific cells are captured and other cells are removed
EasySep Human EpCAM Positive Selection Kit
Stemcell Technologies
Human EpCAM Positive Selection Cocktail
Magnetic Nanoparticles
RoboSep Buffer
EasySep Magnet
ResultsMagnetic capture followed by pressure cycling
Mixed M/F control,
Sperm control
Recovered male DNA
Tools development
• In the order to address the issue of low-copy number
PCR amplification, that may be created by dilution of the
mixed samples, the highly sensitive Miniplex PCR kit
was developed using Qiagen Multiplex PCR Plus Kit
chemistry.
• The kit was based on the previously developed Miniplex
concept for amplification of smaller fragments and was
designed to use the IPCRp method.
• Butler JM, Shen Y, McCord BR. The development of reduced size STR amplifications
as tools for analysis of degraded DNA. J Forensic Sci, 2003;48:1054-64.
• Pero Dimsoski, Sam L. Woo. Increasing Detection of Polymerase Chain Reaction
(PCR) by Isolation of PCR Products (IPCRp). Croat Med J 2005;46(4):619-621.
IPCRp (Isolation of PCR product) Miniplex
Combining the Miniplex with IPCRp
Miniplex IPCRp
THO1FGA
CSFD21
TPOX D7
9947a standard
Miniplex IPCRp sensitivity
0.250ngPCR
0.125ngPCR
0.062ngPCR
0.015 ngPCR
Pressure cycling with touch DNA
Method
•Stainless steel bar wiped with swabs compared to control
buccal swabs
•Qiagen Micro kit aided by pressure cycling was used for
DNA extraction
•Miniplex IPCRp kit used for amplification
Profiles obtained from buccal swabs (top)
and swabbing fingerprint (bottom) w/ Pressure cycling
1 ng in PCR
0.20 ng in PCR
Results
•Full profiles from fingerprints were obtained from
three different individuals (not all results shown)
using modified Miniplex kit.
•The [DNA] for all three individuals was bellow 0.02 ng.
•The extraction included pressure cycling followed by
Qiagen Micro kit.
Obtaining horse profile from
challenging field samples
• Due to exposure of elements and other
factors, it is very challenging to obtain a
DNA profile from feces.
• A sensitive six-plex PCR horse kit was
developed designed to work with
IPCRp method.
6-plex horse kit with IPCRp
Fecal DNA from horse – IPCRp Qiagen Micro kit
(Pressurized extraction not used)
VHL20HTG4
HTG6 HMS6
HTG7 HMS3
Improved Profile
Pressure extraction, Qiagen mini kit, IPCRp
VHL20HTG4
HTG6HMS6
HTG7HMS3
Results
•Partial equine DNA profiles from fecal matter were obtained in
10 out of 27 samples using nested PCR.
•Full profiles were obtained in 17 out of 27 samples by
pressure cycling and IPCRp methods.
•Pressure cycling along with IPCRp improved the likelihood of
obtaining a full equine DNA profile from fecal samples.
Conclusions
• IPCRp + Pressure cycling permits cleaner PCR products
and improved recovery of low level/ inhibited DNA.
Results with touch and equine DNA show improved
recovery.
• Pressure cycling also has demonstrated the capability to
selectively enrich male DNA in mixtures through the use
of DTT and TCEP to disrupt sperm cells.
• By combing pressure cycling and immunomagnetic
capturing of epithelial cells, it is possible to obtain a
cleaner male profile.
Acknowledgements
FIU
Vanessa Martinez
Deepthi Nori
Julian Mendel
Dee Mills
Melissa Villarreal
Stemcell Technologies
Mihael Warnement
National Institute of Justice, Pressure Biosciences, Promega
This project was supported through NIJ grant # 2011-NE-BX-K550. Points of view in the document are those of the authors and do not necessarily represent the official view of the US Department of Justice.