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.