1. Lab Techniques: DNA/Genetics Doug Dluzen Lab Lecture 1 ddluzen@hmc.psu.edu Eom et al. Nature 1996.

2. Overview PCR – Discovery – qRT-PCR – Genotyping Restriction Digests – Gel Electrophoresis – RFLP Western Blotting Southern Blotting Northern Blotting Using Genomic Databases

3. The Polymerase Chain Reaction http://elementy.ru/images/eltpub/kary-mullis.jpgMolecular Diagnostics: For the Clinical Laboratorian, Second Edition -Allows one to replicate, study, and manipulate specific DNA sequences of interest -PCR revolutionized molecular biology -PCR technology is used in almost every lab and hospital

4. Amplifying DNA in Vitro: The Polymerase Chain Reaction (PCR) The polymerase chain reaction, PCR, can produce many copies of a specific target segment of DNA A three-step cycle—heating, cooling, and replication—brings about a chain reaction that produces an exponentially growing population of identical DNA molecules The key to PCR is an unusual, heat-stable DNA polymerase called Taq polymerase. Adopted from Dr. Sairam Lecture Slides

5. Genomic DNA Target sequence 5 5 3 3 Adopted from Dr. Sairam Lecture Slides The Steps of PCR

6. Denaturation Annealing Extension Primers New nucleo- tides Cycle 1 yields 2 molecules 5 5 3 3 2 3 1 Figure 20.8b Adopted from Dr. Sairam Lecture Slides

7. Cycle 2 yields 4 molecules Adopted from Dr. Sairam Lecture Slides

8. Figure 20.8d Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence Adopted from Dr. Sairam Lecture Slides

9. DNA technology allows us to study the sequence, expression, and function of a gene DNA cloning allows researchers to – Compare genes and alleles between individuals – Locate gene expression in a body – Determine the role of a gene in an organism Several techniques are used to analyze the DNA of genes – Already mentioned PCR Adopted from Dr. Sairam Lecture Slides

10. Gel Electrophoresis One indirect method of rapidly analyzing and comparing genomes is gel electrophoresis This technique uses a gel as a molecular sieve to separate nucleic acids or proteins by size, electrical charge, and other properties A current is applied that causes charged molecules to move through the gel Molecules are sorted into “bands” by their size Adopted from Dr. Sairam Lecture Slides

11. Pulsed field gel electrophoresis Adopted from Dr. Sairam Lecture Slides http://academic.brooklyn.cuny.edu/biology/bio4fv/page/molecular%20biology/dsDNA.jpg

12. Mixture of DNA mol- ecules of different sizes Power source Longer molecules Cathode Anode Wells Gel Shorter molecules TECHNIQUE RESULTS 1 2     Adopted from Dr. Sairam Lecture Slides

13. Oh the places You’ll Go Where have we been? – PCR, Electrophoresis Where are we going? – PCR Technologies

14. Technology #1: qRT-PCR Stands for quantitative real-time PCR Allows one to: – Assess gene expression levels – Measure and compare gene expression levels Uses PCR technology and resources with a fluorescent twist – Fluorescent light is used as a marker for gene expression evaluation – The more light = more gene mRNA expression

15. Flourescent (qRT-PCR) http://www.scripps.edu/florida/technologies/genomics/images/TaqManMethod.png The Methodology An Amplifcation Curve: the visualization and data of qRT-PCR

16. Comparing Gene Expression Amplification curves determine the amount of gene mRNA present – One can compare the expression of a gene of interest in two different conditions Normal vs cancerous Stressed vs unstressed Patient #1 vs Patient #2 But! An internal control is needed

17. The Internal qRT-PCR Control Why do we need an internal control? – PCR gene expression analysis actually measures the difference of a gene’s mRNA levels after that same gene’s expression was compared to a house- keeping gene ubiquitously expressed in all conditions

18. The internal qrt-pcr control How does this work? – 1. A gene’s (G.O.I.) mRNA is measured via PCR in Tissue #1 – 2. A control gene’s mRNA is measured via PCR in Tissue #1 – 3. A ratio of the GOI to the control is measured – 4. Steps 1 – 3 occur in Tissue #2 – 5. The ratios of Tissue 1 vs. Tissue 2 are then compared to determine their relative expression This is known as the ∆∆Ct method

19. The Ct value

20. One can use PCR to determine an individual’s Single Nucleotide Polymorphisms (SNPs) TeCh #2 - SNP Genotyping

21. Single Nucleotide Polymorphisms In humans, researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition Genetic markers called SNPs (single nucleotide polymorphisms) occur on average every 100–300 base pairs SNPs can be detected by PCR, and any SNP shared by people affected with a disorder but not among unaffected people may pinpoint the location of the disease-causing gene © 2011 Pearson Educatio, Inc. Adopted from Dr. Sairam Lecture Slides

22. DNA SNP Normal allele Disease-causing allele T C Single Nucleotide Polymorphisms Adopted from Dr. Sairam Lecture Slides

23. SNP Genotyping Probe 1 – Allele A Probe 2– Allele B - The colored output is measured

24. TeCh #2 - SNP Genotyping Wild Type Heterozygote Homozygous

25. SNP Genotyping

26. Tech #3 – Copy Number Variation In addition to SNPs, the human genome is widely variable with respect to gene copy number between any two individuals He et al. Trends in Molecular Medicine. 2011.

27. Copy Number Variation

28. UGT2B17 CNV Taqman CNV assay designed to detect deletion polymorphism in pancreatic gDNA samples – 813 samples, Caucasian Each sample run as the following: – 2x Master mix – 5 uL – 20x CNV assay + 20 x reference assay (RNaseP) – 1 uL – 10 ng gDNA – 1 uL – Water – 3 uL 10 uL total

29. UGT2B17 Copy Number Variation – An Example

30. Analyzing DNA

31. Using Restriction Enzymes to Make Recombinant DNA Bacterial restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites A restriction enzyme usually makes many cuts, yielding restriction fragments The most useful restriction enzymes cut DNA in a staggered way, producing fragments with “sticky ends.” Adopted from Dr. Sairam Lecture Slides

32. Restriction Digest Sites Adopted from Dr. Sairam Lecture Slides

33. DNA Ligase Sticky ends can bond with complementary sticky ends of other fragments DNA ligase is an enzyme that seals the bonds between restriction fragments Adopted from Dr. Sairam Lecture Slides

34. Recombinant DNA molecule One possible combination DNA ligase seals strands DNA fragment added from another molecule cut by same enzyme. Base pairing occurs. Restriction enzyme cuts sugar-phosphate backbones. Restriction site DNA 5 5 5 5 5 5 5 5 55 5 5 55 5 5 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 231 Sticky end GAATTC CTTAAG CTTAA G AATTC G G G CTTAA G G G G AATT C C TTAA Adopted from Dr. Sairam Lecture Slides

35. Using Restriction Enzymes In restriction fragment analysis, DNA fragments produced by restriction enzyme digestion of a DNA molecule are sorted by gel electrophoresis Restriction fragment analysis can be used to compare two different DNA molecules, such as two alleles for a gene if the nucleotide difference alters a restriction site Sequence changes that alter restriction sites are called RFLPs (restriction fragment length polymorphisms) Adopted from Dr. Sairam Lecture Slides

36. Normal  -globin allele Sickle-cell mutant  -globin allele Large fragment Normal allele Sickle-cell allele 201 bp 175 bp 376 bp (a) Dde I restriction sites in normal and sickle-cell alleles of the  -globin gene (b) Electrophoresis of restriction fragments from normal and sickle-cell alleles 201 bp 175 bp 376 bp Large fragment Dde I RFLP Analysis Adopted from Dr. Sairam Lecture Slides

37. Restriction Digests Need to check if PCR fragment, generally cloned into a useful reporter, has the correct orientation – DNA inserts can ligate into a plasmid in two directions

38. Detection of Specific DNA and Protein Sequences - Blotting Whether you are analyzing DNA, RNA, or protein the underlying principle is the same Use of a probe that signals presence/absence of a gene or protein of interest Labeled (chemically, radioactively, etc.)probe is used to visualize your target

39. A technique called Southern blotting combines gel electrophoresis of DNA fragments with nucleic acid hybridization Specific DNA fragments can be identified by Southern blotting, using labeled probes that hybridize to the DNA immobilized on a “blot” of gel Southern Blotting Adopted from Dr. Sairam Lecture Slides

40. Southern Blotting Adopted from http://homepages.strath.ac.uk/~dfs99109/BB211/RecombDNAtechlect2.html#northerns

41. Southern Blotting A southern blot can distinguish: 1.The presence of a particular gene of interest 2.Number of copies of that gene 3.Genomic rearrangements 4.Mutations of restriction digest sites -Southern blots are very sensitive Adopted from http://homepages.strath.ac.uk/~dfs99109/BB211/RecombDNAtechlect2.html#northerns

42. Northern Blotting Northern blotting combines gel electrophoresis of mRNA followed by hybridization with a probe on a membrane Identification of mRNA at a particular developmental stage suggests protein function at that stage Adopted from http://homepages.strath.ac.uk/~dfs99109/BB211/RecombDNAtechlect2.html#northerns Adopted from Dr. Sairam Lecture Slides

43. Northern Blotting Same principle as southern blotting, except RNA is measured as opposed to DNA – RNA can also bind to nitrocellulose membrane – Uses formaldehyde as a denaturing reagent Used to identify tissue and temporal expression of a particular gene – Sensitive – Used to measure expression levels of particular mRNA

44. Western Blotting Powerful tool to detect presence and expression levels of a particular protein – Use of an antibody – specific protein molecule will bind to specific protein sequence on the protein of interest This specific protein sequence is called an epitope As with northern and southern blotting, proteins are sorted by molecular weight, transferred to a membrane, and probed – Protein presence, expression, and quantity can be measured

45. Western Blotting - Principles Adopted from GE Healthcare: Western Blotting Principles

46. Western Blotting Methods 1. Electrophoresis – Denaturing Gel Adopted from GE Healthcare: Western Blotting Principles

47. Western Blot Methods 2. Transfer from gel to membrane Adopted from GE Healthcare: Western Blotting Principles

48. Western Blot Methods Once protein transferred to membrane – Incubate in protein buffer (generally 5% milk solution) to bind all regions of blot not bound by transferred protein Incubate with primary and secondary antibodies Visualize!

49. And now for something completely different…

50. Browsing the Genome Browsers NCBI - http://www.ncbi.nlm.nih.gov/ Ensembl - http://useast.ensembl.org/index.html University of California Santa Cruz - http://genome.ucsc.edu/ Pubmed - http://www.ncbi.nlm.nih.gov/pubmed/

51. NCBI

52. BLAST!

53. Ensembl

54. Ensembl – THE MANY USES

55. I see U see UCSC

56. UCSC

57. Other Useful Odds and Ends HapMap Project – encyclopedia of human and mouse SNP variation and genotype frequencies www.hapmap.org TargetScan – microRNA prediction Pubmed – uses NCBI database for literature searches, protein and nucleotide sequences

58. Thank you! Questions? ddluzen@hmc.psu.edu