Bootcamp: Molecular Biology T echniques and I nterpretation

1. Bootcamp: Molecular Biology Techniques and Interpretation Winter 2013-2014

2. Today’s outline • Manipulating DNA: • Enzyme toolkit • Cloning • cDNA • Libraries • Sequencing • Databases Detecting and quantifying nucleic acids: • Basic nucleic acid properties • Hybridization • Probe design • In situ hybridization • Report genes • Gel electrophoresis • Blotting • PCR • qPCR

3. Nucleic Acid Structure RNA differences from DNA ‐RNA is single stranded ‐RNA contains 2’ –OH, making it more susceptible to hydrolysis ‐RNA uses Uracil instead of Thymine (missing methyl substituent) G‐C interactions are stronger than A‐T interactions (more H‐Bonds)

4. Effect of salt, temperature and pH on nucleic acid duplex formation *Equilibrium of interactions* hydrogen-bonding, pi-pi interactions vs. charge repulsion Salt More salt screens backbone charges decreasing strand repulsion Temperature Higher temperature causes separation of the DNA strands or ‘melting’ pH Alkaline and acidic pH can both denature DNA, and acidic pH promotes depurination.

5. Hybridization Higher temperature requires higher degree of complementarity Alberts et al., Molecular Biology of the Cell

6. Designing a hybridization probe • 2 key features • Length • Complementarity • What sort of questions can you answer with these probes? • How specific do you want your probe to be? • Do you want to pick up highly similar sequences? Or more dissimilar sequences?

7. In situ hybridization (ISH)

8. In situ hybridization (ISH) Probe consists of complimentary RNA bound to digoxigenin, or DIG for short

9. In situ hybridization (ISH) Probe consists of complimentary RNA bound to digoxigenin, or DIG for short Anti‐DIG antibody (binds selectively to DIG) Alkaline phosphatase (AP) is covalently bonded to antibody

10. In situ hybridization (ISH) Probe consists of complimentary RNA bound to digoxigenin, or DIG for short BCIP and NBT Anti‐DIG antibody (binds selectively to DIG) Alkaline phosphatase (AP) is covalently bonded to antibody The color blue (BCIP‐NBT heterodimer)

11. Gel Electrophoresis Nucleic Acids: Proteins:

12. Blotting: Detecting presence of specific DNA/RNA sequences using probes Southern = DNA Northern = RNA

13. Blotting: Detecting the presence of specific proteins Western = Protein

14. Problems with Blotting - Need lots of material to be visible 1 cell 1‐15pg mRNA - Poor resolution 1 cell →1‐15pg mRNA 1 lane →5‐10ug mRNA How many cells would be required for a lane? 3M-4M cells ‐ Gel lanes fill and blur quite quickly ‐ Relative quantification of material generally can only occur above 2 orders of magnitude

15. Polymerase Chain Reaction (PCR) Reactionmixturecontains: DNA Polymerase 2 Primers dNTP (DNA nucleotides) MgCl2 Double stranded DNA Template PCR reactions are performed on a programmable thermocycling heat block

16. How to choose/design a PCR primer PCR primers should be unique to the region you intend to amplify • Length (optimal length 18‐22 bp) • Sequence Complementarity to the ends of your target DNA • Melting temperature (Tm) • Primers shouldn’t form secondary structure

17. Primer design For amplification, a direct match: One can also easily make mutations/insertions/deletions to DNA using the primer:

18. Quantitative PCR (qPCR) 100 to 10,000 times more sensitive than Blotting The principle In the semi‐quantitative PCR method, a set number of cycles of PCR amplification were run. The product is run out on a gel and if multiple species are amplified, probe for the correct sequence. Real Time‐PCR (aka qPCR) gives cycle by cycle analyses of all species levels as above leading to a much larger dynamic range (~5 orders of magnitude) and a much higher calculation accuracy of the original copy number. (There is no one cycle number above where all species are in linear growth phase)

19. Detecting and quantifying specific nucleic acid sequences rely on hybridization • Probe design for in situ hybridization and blotting • Primer design for PCR qPCR can quantitate gene expression levels while requiring a lot less sample compared to a Northern blot • However, PCR primers are short and need to be very specific (high complementarity). Blotting probes are often very long, and do not always need 100% complementarity. This allows blotting to detect certain things that PCR cannot. Can you think of any examples?

20. Enzyme toolkit for manipulating DNA • DNA polymerase is used to copy a DNA strand from a complementary template • Restriction Enzymes cut DNA at specific recognition sequences • Ligases join DNA sequences together • “Destruction” Nucleases • S1 and DNaseI are commonly used nuclease to degrade DNA • RNAseA is a commonly used nuclease to degrade RNA

21. Restriction Enzymes and Ligases Restriction enzymes Ligases

22. Cloning • What is Cloning? • Process of introducing foreign DNA into a host cell • Recombinant DNA = gene which is inserted into a vector • Vector is a circular piece of DNA which transports a gene into a host cell • A host cell containing inserted gene = a clone • Why do Cloning? • Molecular analysis of genes and their function depends on the isolation and amplification of specific segments of DNA: • Sequencing • Expression of genes, proteins to affect biology pathways • Manipulation

23. Cloning Procedure • Insert cleaved DNA fragments into a plasmid vector • Allows replication within host organism (E. coli) • manipulate DNA • store it in the lab • introduce DNA into organism or cell • generate a library of DNA molecules • Vector characteristics: • Origin of replication • Selectable marker • Restriction enzyme sites

24. Cloning as engineering If you know the gene sequence: • Design primers • Extract genomic DNA for template DNA • Amplify the gene by PCR • You can use PCR methods to modify gene • Ligate (cut and paste) the gene into vector • Transform vectors into bacteria to isolate and purify DNA • Screen colonies carrying the gene and verify by sequencing • Use the plasmid to make a transgenic animal to express modified gene

25. Reporter genes Reporter genes can be cloned into a plasmid and transfected into cells, forcing them to express the protein under the right conditions.

26. From class, we saw that in situhybridization combined with a reporter gene assay can be used to determine the regulatory activity of the stripe 2 module of the Eve gene.

27. Creating Libraries Genomic library • Constructed by ligating fragments of genomic DNA into vectors; each plasmid contains a part of the entire genome cDNA library • Constructed by ligating only coding regions into vectors A DNA library is a population of identical vectors or linear DNA that each carries a different insert Molecular Biology of the Gene 5th ed

28. How does one make a cDNA library? RNA cDNA reverse transcriptase • mRNA isolated from an organism/tissue/cell directly encodes a gene and can be converted to DNA using a reverse transcriptase. The product is called complementary DNA (cDNA).

29. cDNA Alberts et al., Molecular Biology of the Cell

30. Cloning for gene discovery • If you do not know the exact gene sequence: • Isolate the DNA fragment that demonstrates your desired activity by testing a library of genes • If the gene is a homolog: • Use degenerate probe to hybridize to related/similar sequences in the genome

31. dideoxynucleotides (ddNTPs) cause chain termination Molecular Biology of the Gene 5th ed

32. Chain-termination Sanger sequencing

33. UCSC Genome Browser http://genome.ucsc.edu/ PUBMED (via Caltech) http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?otool=caitlib