The Genomics Laboratory

1. The Genomics Laboratory • Key Technologies • PCR • Gel electrophoresis • SNP genotyping through Microarrays • Inputs • Biological samples • Subject and pedigree information • Outputs • Genotypes (raw data) • Report phenotypes (presence of genetic defect) • Report parentage (or predictions of parentage) • Report predictions of other traits

2. Polymerase Chain Reaction • PCR - What is it? • PCR is a laboratory technique for copying a portion of a DNA molecule • PCR can be thought of as DNA replication in-vitro (in a tube) rather than in-vivo (in a cell / living system) • Characteristics • Powerful / Sensitive • Possible to use even in cases where there is a very small amount of DNA available • Theoretically, with even one molecule of DNA, enough copies can be made for scientific analysis • Enabled the biotechnology revolution

3. Why do we want to copy or "amplify" DNA? • Deciphering DNA • Instrumentation capable of "reading" a DNA sequence need a sufficient concentration of DNA to operate • This includes determining the nucleotide present at specific locations in the genome • Can be combined with other techniques such as tagging sequences with labels • Some applications: testing for genetic defects, parentage, viruses, tissue typing, forensics

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

5. Some History...Kary Mullis • Invented 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 position • The 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 molecules • Known for some unconventional ideas, both before and since the invention of PCR • The 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 employer • Cetus later sold the PCR patent to LaRoche for $300M

6. Background: DNA characteristics important to PCR • For replication occur, the DNA strands must separate ("denature" or "melt") • Hydrogen bonds between nucleotides much weaker than covalent bonds on the backbone • Affinity for each nucleotide for its complement • Affinity for a sequence to bind to its complement (and only its complement) • Replication requires a starting place: a primer • Let's review the structure of DNA and how it replicates

7. DNA Components • Nitrogenous 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’ carbon Phosphate covalently bonds with 5’ carbon Normal ribose (OH on 2’ carbon) – RNA deoxyribose (H on 2’ carbon) – DNA dideoxyribose (H on 2’ & 3’ carbon) – used in DNA sequencing • Phosphate: negatively charged credit: from bioalgorithms slide 90

8. Basic DNA Structure Sugar Phosphate Base (A,T, C or G) http://www.bio.miami.edu/dana/104/DNA2.jpg

9. DNA Replication from bioalgorithms slide 98

10. DNA Replication

11. PCR Process • PCR similar to in-vivo DNA replication, with the following differences:

12. PCR Reagents • DNA template • Primers (oligonucleotides) • Important: Must be designed to span fragment of interest, on opposite strands • Taq polymerase • dNTP's • deoxyribonucleoside triphosphates • PCR buffer • w/salts • Magnesium chloride • Water • Combine into a "Master Mix" • Shaken, not stirred

13. PCR Process in a Nutshell • Design oligonucleotide primers that span region to be copied • Add primers to reaction mixture • Thermally 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

14. Taq Polymerase Thermus aquaticus, a thermophilic bacteriadiscovered 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 process • If regular DNA polymerase is used, it is necessary to add it at certain points as it will not tolerate the high temperatures • Use of “taq” makes process much faster

15. Oligonucleotide primers • To 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 place • The term “oligonucleotide” means that the nucleotide was synthesized • In PCR, the oligonucleotide is used as a primer • Note that the two primers are different, one for each DNA strand, designed to bond at opposite ends of the region of interest • This design is important for producing sequences of a precise “unit” length • Do you want to order some primers? Go to the website for Integrated DNA technologies (for example) and specify the desired sequence…they can be ready in a few days!!!

16. PCR Reaction Step 1: Denaturation Raise temperature to 94oC to separate the duplex form of DNA into single strands

17. Step 2: Annealing • Anneal primers at 50-65oC

18. Step 3: Extension • Extend primers: raise temp to 72oC, allowing Taq pol to attach at each priming site and extend a new DNA strand

19. Repeat • Repeat the Denature, Anneal, Extension steps at their respective temperatures…

20. Polymerase Chain Reaction • Problem: Modern instrumentation cannot easily detect single molecules of DNA, making amplification a prerequisite for further analysis • Solution: PCR doubles the number of DNA fragments at every iteration 1… 2… 4… 8…

21. Question 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 really • We’ll take a closer look.

23. Exponential increase in copies of target DNA (well, almost!) See http://www.youtube.com/watch?v=_YgXcJ4n-kQ

24. How is this automated? • Through the “PCR Machine”, known as a Thermal Cycler • Here is a picture: (search YouTube for “PCR Song”!)

25. Single Nucleotide Polymorphism (SNP) Genotyping • ~99% of human genomic loci are identical for all individuals • The remaining 1% appear to account for the vast genetic variation we observe, including in the susceptibility of individuals to disease • A SNP is a specific locus on the genome where there is variation in a significant portion of the population • Variation at a single base pair location • Since each individual inherits one copy of DNA from each parent, each SNP can have three allelic values, commonly referred to as AA, BB, or AB

26. SNP Genotyping Methodology • Use PCR or other (perhaps similar) biochemical method to amplify DNA • Use chemically labeled oligonucleotide primers called “probes” to bind around or in proximity to the SNP • The 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 instrumentation • Example labels are based on detection by florescence or by unique mass

27. Use of dideoxynucleotide (ddNTP) • Dideoxynucleotide (ddNTPs) can be used to extend a probe, to add a base at the SNP position • A ddNTP lacks an –OH (hydroxyl) at the 3’ position on the deoxyribose sugar, preventing addition of additional bases, thus terminating the chain

28. ddNTPs – Buy them labeled!

29. Single base extension of probe in the Illumina “Infinium” asay

30. 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 field • The size of DNA fragments can then be determined by comparing their migration in the gel to known size standards.

31. Gel Electrophoresis

32. SNP Genotyping with Gel Electrophoresis

33. Gel Electrophoresis (continued)

35. Sequenom iPlex “MassARRAY” • Will use the Sequenom iPlex as an example of Microarray technology for SNP genotyping • Many thanks go to Dr. Adam Shahid (Molecular Biologist) of Pfizer Animal Genetics for describing this technology and process • Many 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

36. Sequenom iPlex – Assay Design • Develop assay to isolate and amplify DNA sequences called “probes” that are terminated by the SNPs of interest • The assay design will require that each SNP-terminated probe have a distinct mass (within the experiment). • Mass-modified ddNTPs are used to facilitate this • Using the MALDI-TOF mass spectrometer (+ software), the SNP allele can be accurately identified based on mass

37. Examples using “10-mer” sequences • Example that will not work (masses not unque) • Sequence ACGATCGAAC precedes SNP-X • Sequence ACGATCGAAT precedes SNP-Y • Note that the first 9 bases are identical between these two sequences • In 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 C • Mass (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 length • Trivial example, but becomes more complex when considering many SNPs being assayed at once and taking instrument tolerances into consideration

38. More on Oligonucleotides • Sequenom’sMassARRAY Designer software automatically designs PCR and extension primers (probes) for each SNP to be investigated • Can be somewhat computationally intensive! • Required oligonucleotides then are ordered from suppliers

39. MALDI-TOF Mass Spectrometry • Allows 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-TOF • Crystal is vaporized by a laser and analyte (extended oligo) is ionized and “flies” to the oppositely charged end of the ionization chamber • Masses are individually detected based on flight time • Software translates masses into SNP allele readouts

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

42. Example SNP Genotype Data SNP Name Sample ID Allele1 Allele2 ARS-BFGL-BAC-10975 US4042705 A A ARS-BFGL-BAC-11025 US4042706 A B ARS-BFGL-BAC-11044 US4042707 B B

43. 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.html • “The 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.php • “PCR”, http://www.youtube.com/watch?v=_YgXcJ4n-kQ • “The PCR Song by Scientists for Better PCR”, http://www.youtube.com/watch?v=7uafUVNkuzg&feature=related • LaFramboise, 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 MassARRAYiPLEX Platform”, http://jmgroup.pl/kawaska/download/SNP%20Genotyping%20Using%20the%20Sequenom.pdf • Chan, Michael, “Application of PCR and Microarray in Molecular Biology”, www.slideworld.org • Shahid, Adam, Ph. D., Pfizer Animal Genetics, one hour interview on PCR, July 11, 2012 • Shahid, Adam, Ph. D., Pfizer Animal Genetics, one hour interview on SNP genotyping, July 12, 2012