Presentation #1 Proposal, Tom Klomparens, CS 6030 Title: Introduction to the Genomics Laboratory

The Genomics LaboratoryKey TechnologiesPCRGel electrophoresisSNP genotyping through MicroarraysInputsBiological samplesSubject and pedigree informationOutputsGenotypes (raw data)Report phenotypes (presence of genetic defect)Report parentage (or predictions of parentage)Report predictions of other traits

Polymerase Chain ReactionPCR - What is it?PCR is a laboratory technique for copying a portion of a DNA moleculePCR can be thought of as DNA replication in-vitro (in a tube) rather than in-vivo (in a cell / living system)CharacteristicsPowerful / SensitivePossible to use even in cases where there is a very small amount of DNA availableTheoretically, with even one molecule of DNA, enough copies can be made for scientific analysisEnabled the biotechnology revolution

Why do we want to copy or "amplify" DNA?Deciphering DNAInstrumentation capable of "reading" a DNA sequence need a sufficient concentration of DNA to operateThis includes determining the nucleotide present at specific locations in the genomeCan be combined with other techniques such as tagging sequences with labelsSome applications: testing for genetic defects, parentage, viruses, tissue typing, forensics

Why do we want to copy or "amplify" DNA? (2)Building sequencesSequences can be designed and constructed -- PCR provides raw materialPCR provides a means of selectively copying sequences of interest -- such as genesCombined with other biochemical methods, part of bioengineering "toolkit"Resulting sequences can be further manipulated to create "recombinant DNA" (rDNA) and even inserted into a genomeSome applications that are enabled: measurement of gene expression, production of recombinant protein products such as insulin and HGH, genetically engineered crops

Some History...Kary MullisInvented in 1983 by Kary Mullis while driving his Honda Civic on the Pacific Coast Highway 128 from San Francisco to Mendocino"I do my best thinking while driving"He was working out in his mind, as he drove, a process for detecting the nucleotide at a specific DNA positionThe process he was contemplating reminded me of the SNP genotyping process that I'll be discussing later today, except that he was working with very few DNA moleculesKnown for some unconventional ideas, both before and since the invention of PCRThe invention of PCR revolutionized molecular biology, and made possible the sequencing of the human genome (and others)Yet the idea is of such "utter simplicity" that the Mullis indicated the initial response of most scientists is: "Why didn't I think of that?"Mullis received the Nobel Prize in Chemistry in 1993, and a $10,000 bonus from Cetus, his employerCetus later sold the PCR patent to LaRoche for $300M

Background: DNA characteristics important to PCRFor replication occur, the DNA strands must separate ("denature" or "melt")Hydrogen bonds between nucleotides much weaker than covalent bonds on the backboneAffinity for each nucleotide for its complementAffinity for a sequence to bind to its complement (and only its complement)Replication requires a starting place: a primerLet's review the structure of DNA and how it replicates

DNA ComponentsNitrogenous Base: N is important for hydrogen bonding between bases A adenine with T thymine (double H-bond)C cytosine with G guanine (triple H-bond)

Sugar: Ribose (5 carbon)Base covalently bonds with 1 carbonPhosphate covalently bonds with 5 carbonNormal ribose (OH on 2 carbon) RNAdeoxyribose (H on 2 carbon) DNA dideoxyribose (H on 2 & 3 carbon) used in DNA sequencing

Phosphate:negatively chargedcredit: from bioalgorithms slide 90Basic DNA Structure

PhosphateBase (A,T, C or G)http://www.bio.miami.edu/dana/104/DNA2.jpgSugarDNA Replication

from bioalgorithms slide 989DNA Replication

PCR ProcessPCR similar to in-vivo DNA replication, with the following differences:

Replication StepIn-vivoPCRPCR dependent onMelting of DNA strandsInitiated by enzymes, especially helicaseIncrease temperatureWeak hydrogen bonds that can be broken with moderate heat, tolerance of in-vitro system to heatSynthesis of primerDNA primaseOligonucleotides synthesized in the laboratoryTechnology to synthesize "oligos"Addition of nucleotides ("elongation")DNA polymeraseTaq polymeraseDiscovery of "taq", an enzyme found in bacteria living only in hot springsPCR ReagentsDNA templatePrimers (oligonucleotides)Important: Must be designed to span fragment of interest, on opposite strandsTaq polymerasedNTP'sdeoxyribonucleoside triphosphatesPCR bufferw/saltsMagnesium chlorideWaterCombine into a "Master Mix"Shaken, not stirred

PCR Process in a NutshellDesign oligonucleotide primers that span region to be copiedAdd primers to reaction mixtureThermally denature the target DNA (94 deg-C)Reduce temperature to allow annealing of primers and target DNA (60 deg-C)Increase temp. for efficient elongation (72 deg-C)Repeat steps 3-5 for 25-35 cycles

Taq PolymeraseThermus aquaticus, a thermophilic bacteria discovered in 1969 in hot spring of Yellowstone National park . It can tolerate high temperature. The DNA polymerase (Taq polymerase) was isolated.Taq polymerase was an innovation added subsequent to original invention of PCR processIf regular DNA polymerase is used, it is necessary to add it at certain points as it will not tolerate the high temperaturesUse of taq makes process much faster

Oligonucleotide primersTo perform PCR, 10-20bp sequences on both sides of the sequence to be amplified must be known because DNA (taq) polymerase requires a primer to synthesize a new strand of DNA

The primer must be designed to be very specific to the sequence, so that it will only bind in one placeThe term oligonucleotide means that the nucleotide was synthesizedIn PCR, the oligonucleotide is used as a primerNote that the two primers are different, one for each DNA strand, designed to bond at opposite ends of the region of interestThis design is important for producing sequences of a precise unit lengthDo you want to order some primers? Go to the website for Integrated DNA technologies (for example) and specify the desired sequencethey can be ready in a few days!!!PCR Reaction Step 1: Denaturation

Raise temperature to 94oC to separate the duplex form of DNA into single strandsStep 2: Annealing

Anneal primers at 50-65oCStep 3: Extension

Extend primers: raise temp to 72oC, allowing Taq pol to attach at each priming site and extend a new DNA strand

RepeatRepeat the Denature, Anneal, Extension steps at their respective temperatures

Polymerase Chain ReactionProblem: Modern instrumentation cannot easily detect single molecules of DNA, making amplification a prerequisite for further analysisSolution: PCR doubles the number of DNA fragments at every iteration

1 2 4 8Question that was troubling (to me):How does the PCR process control the length of the copied DNA fragments?By the amount of time the process is allowed to stay at the Extension temperature? -Not reallyWell take a closer look.

Exponential increase in copies of target DNA (well, almost!)Cycle NumberTarget DNA CopiesLonger CopiesTotal Copies10222044326848816522103230 1,073,741,766 60 1,073,741,826 See http://www.youtube.com/watch?v=_YgXcJ4n-kQHow is this automated?Through the PCR Machine, known as a Thermal CyclerHere is a picture: (search YouTube for PCR Song!)

Single Nucleotide Polymorphism (SNP) Genotyping~99% of human genomic loci are identical for all individualsThe remaining 1% appear to account for the vast genetic variation we observe, including in the susceptibility of individuals to diseaseA SNP is a specific locus on the genome where there is variation in a significant portion of the populationVariation at a single base pair locationSince each individual inherits one copy of DNA from each parent, each SNP can have three allelic values, commonly referred to as AA, BB, or ABSNP Genotyping MethodologyUse PCR or other (perhaps similar) biochemical method to amplify DNAUse chemically labeled oligonucleotide primers called probes to bind around or in proximity to the SNPThe label may also be attached at the SNP position (discussed in next slides)The purpose of the label is to allow detection of the presence of the SNP through instrumentationExample labels are based on detection by florescence or by unique massUse of dideoxynucleotide (ddNTP)Dideoxynucleotide (ddNTPs) can be used to extend a probe, to add a base at the SNP positionA ddNTP lacks an OH (hydroxyl) at the 3 position on the deoxyribose sugar, preventing addition of additional bases, thus terminating the chain

ddNTPs Buy them labeled!

Single base extension of probe in the Illumina Infinium asay

Electrophoresis

A copolymer of mannose and galactose, agaraose, when melted and recooled, forms a gel with pores sizes dependent upon the concentration of agarose

The phosphate backbone of DNA is highly negatively charged, therefore DNA will migrate in an electric fieldThe size of DNA fragments can then be determined by comparing their migration in the gel to known size standards.

Gel ElectrophoresisSNP Genotyping with Gel Electrophoresis

Gel Electrophoresis (continued)

Sequenom iPlex MassARRAYWill use the Sequenom iPlex as an example of Microarray technology for SNP genotypingMany thanks go to Dr. Adam Shahid (Molecular Biologist) of Pfizer Animal Genetics for describing this technology and processMany DNA microarray platforms have the assays and chemistry pre-packaged and ready to detect specific SNPs (or other DNA or RNA of interest)The Sequenom is useful as a general SNP genotyping machine, allowing development of assays for specific SNPs of interest

Sequenom iPlex Assay DesignDevelop assay to isolate and amplify DNA sequences called probes that are terminated by the SNPs of interestThe assay design will require that each SNP-terminated probe have a distinct mass (within the experiment).Mass-modified ddNTPs are used to facilitate thisUsing the MALDI-TOF mass spectrometer (+ software), the SNP allele can be accurately identified based on mass

Examples using 10-mer sequencesExample that will not work (masses not unque)Sequence ACGATCGAAC precedes SNP-XSequence ACGATCGAAT precedes SNP-YNote that the first 9 bases are identical between these two sequencesIn this example, the probes for SNP-X and SNP-Y would have the same mass if SNP-X had an allele of T and SNP-Y had an allele CMass (ACGATCGAAC T) = Mass (ACGATCGAATC)(i.e., since they contain the same 11 bases)This would be easily solved if the two binding sequences were of different lengthTrivial example, but becomes more complex when considering many SNPs being assayed at once and taking instrument tolerances into considerationMore on OligonucleotidesSequenoms MassARRAY Designer software automatically designs PCR and extension primers (probes) for each SNP to be investigatedCan be somewhat computationally intensive!Required oligonucleotides then are ordered from suppliersMALDI-TOF Mass SpectrometryAllows molecular mass readout of extended oligonucleotides (i.e., probes bound to SNPs of interest)Extended oligos are crystalized in special matrix on a silica chip (via robotic liquid handling system), and chip is loaded into the MALDI-TOFCrystal is vaporized by a laser and analyte (extended oligo) is ionized and flies to the oppositely charged end of the ionization chamberMasses are individually detected based on flight timeSoftware translates masses into SNP allele readouts

The Sequenom Process (iPlex)Use PCR to amplify DNA fragments containing SNPsUse shrimp alkaline phosphatase (SAP) to neutralize unincorporated dNTPs (dephosphorylation)Extension reaction add ddNTPsResin step (de-salting)Spotting, loading, and running on MALDI-TOF mass spectrometer

Example SNP Genotype DataSNP NameSample IDAllele1 Allele2 ARS-BFGL-BAC-10975US4042705AAARS-BFGL-BAC-11025US4042706ABARS-BFGL-BAC-11044US4042707BB

Presentation #1 Tom Klomparens, CS 6030Title: Introduction to the Genomics Laboratory

References:Mullis, K. B., "The Unusual Origin of the Polymerase Chain Reaction", Scientific American, April 1990. Mullis, K. B., Nobel Prize lecture, http://www.nobelprize.org/nobel_prizes/chemistry/laureates/1993/mullis-lecture.htmlThe Invention of PCR, at bitesizebio.com, http://bitesizebio.com/articles/the-invention-of-pcr/Russell, Peter J., iGenetics, A Molecular Approach, chapter 8 (The Mapping and Sequencing of Genomes) and chapter 9 (Functional and Comparative Genomics)Hoppe, Pamela (WMU), lecture slides from BIOS 2500 (General Genetics)Jones, Neil C., and Pevzner, Pavel A., An Introduction to Bioinformatics Algorithms, chapter 3, and Molecular Biology slides at http://bix.ucsd.edu/bioalgorithms/slides.phpPCR, http://www.youtube.com/watch?v=_YgXcJ4n-kQThe PCR Song by Scientists for Better PCR, http://www.youtube.com/watch?v=7uafUVNkuzg&feature=relatedLaFramboise, Thomas, et. al., SURVEY and SUMMARY: Single nucleotide polymorphism arrays: a decade of biological, computational and technlogical advances, Nucleic Acides Research, 2009, Vol, 37, No 13 (4181-4193)Gabriel, Stacey, et. al., SNP Genotyping Using the Sequenom MassARRAY iPLEX Platform, http://jmgroup.pl/kawaska/download/SNP%20Genotyping%20Using%20the%20Sequenom.pdfChan, Michael, Application of PCR and Microarray in Molecular Biology, www.slideworld.orgShahid, Adam, Ph. D., Pfizer Animal Genetics, one hour interview on PCR, July 11, 2012Shahid, Adam, Ph. D., Pfizer Animal Genetics, one hour interview on SNP genotyping, July 12, 2012