ABSTRACT

1
ABSTRACT

• The aim of this project is to identify sets of
  dinucleotide repeats in the human genome that
  would be informative enough for forensic and
  other diagnostic applications. Since screening
  for such repeats would be difficult with
  conventional gel electrophoretic methods, non
  radioactive Matrix Assisted Laser
  Desorption/Ionization-Time of Flight (MALDI-TOF)
  mass spectrometric method will be employed. Basic
  methodology includes polymerase chain reaction
  (PCR) amplification of the target sequences from
  a panel of human DNAs and analysis of the
  products by gel electrophoresis and mass
  spectrometry. This would provide a rapid
  non-radioactive method for analysis of Short
  Tandem Repeats (STRs) for forensic and other
  diagnostic applications.

2
INTRODUCTION

• Numerous regions of the human genome contain
  a variable number of STRs that can be amplified
  using PCR. Though these repeats have a high
  frequency of inter-individual variation, they are
  currently not being used for diagnostic purposes
  as it is very difficult to resolve two base
  differences by gel electrophoresis using
  non-radioactive methods. Mass spectrometry offers
  an alternative non-radioactive and more accurate
  detection method with high resolving power. While
  the use of a gel at a crime scene is limited to
  DNA analysis, MALDI can be used for serum protein
  analysis for typing, drug testing and chemical
  analysis of blood samples as well as DNA
  analysis. A compilation of dinucleotide repeats
  using MALDI-TOF MS, will thus provide a
  fingerprint unique to an individual which can be
  used for forensic and diagnostic applications.

3
PURPOSE/APPLICATION

• To analyze dinucleotide repeats in the human
  genome
• To develop a set of STR markers for diagnostic
  and forensic applications using MALDI-TOF MS

4
Polymerase Chain Reaction
5
MATERIALS AND METHODS

• 1. DNA amplification by PCR using Perkin Elmer
  Thermal Cycler
• 2. Agarose gel electrophoresis (stained with
  Ethidium bromide)
• 3. Purification of DNA using phenolchloroform
  extraction
• 4. Use of denaturing acrylamides for molecular
  weight determination
• 5.Acrylamide gel electrophoresis (stained with
  SYBRTM(FMC) green)
• 6. Purification using ZipTip/ QIAgen and
  Precipitation using EtOH
• 7. Matrix preparation
• 8. Analysis using MALDI-TOF MS

6
RESULTS

• Sixteen primers from the original set of
  fifty were selected for further testing. PCR
  product analysis with silver staining and
  sequencing gels have been inadequate as too much
  background develops before the PCR product can be
  visualized. However, PCR product analysis using
  polyacrylamide gels stained with SYBRTM (FMC)
  green has shown high concentrations with no
  background. These gels were then used to further
  determine concentrations using several templates
  against one primer. Furthermore, multiple bands
  developed on these gels indicating DNA
  variability.

7
Original set of 5O Primers analyzed byAgarose
gel electrophoresis
8
Mass Spectra
100 bp
200 bp
150 bp
9
Acrylamide gels stained with SYBRTM(FMC) green
Primers 29 34 36 37 38 43 45 49

• 222
• 179
• 126
• 75
• 65
• 51
• 36
• Pgem Marker

10
FUTURE WORK

• Although all 16 chosen primers have been
  tested extensively, there is still a need to
  carry out further gel analysis to determine the
  combinations of primers, templates and polymerase
  that produce the optimal product concentrations.
  Once this is achieved, DNA fragments amplified by
  PCR will be analyzed using MALDI-TOF MS such that
  two base pair differences can be resolved.

11
ACKNOWLEDGEMENTS

• I would like to thank ORISE for this opportunity
  to conduct research at ORNL.
• I would especially like to thank Winston Chen,
  Lauri Sammartano, Narayana Isola, Steve Allman
  and Darlene Holt for all their help and guidance.