1. 1 Cloning and Sequencing Explorer Series 鲁林荣 娄 军 方 瑜

2. Related knowledge Molecular cloning Plasmid/vector DNA sequencing technology DNA sequence analysis

3. Molecular Cloning Overview Cloning refers to the production of multiple copies. Molecular cloning is the process of of making multiple copies of a molecule. Gene cloning is a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules which can replicate and expand within host organisms.

4. Plasmid Plasmid: Extrachromosomal genetic element also made of a circular DNA molecule.

5. Clone Selection 1. Selection for Plasmd: Host cell lack Ampicilin resistant gene Amp r and cannot grow without the introduced plasmid in media with the antibiotic. 2. Selection for inserted gene fragment: Plasmid express lac Z gene which is disrupted by the insertion of DNA fragment.

6. Cloning Vectors: 1.2000 to 10000bp in length; 2.Self replicate in bacteria 3.High copy numbers 4.Help the servive of host for selection (antibiotic resistant gene) 5.Multiple cloning sites 6.White and Blue selection (lacZ) 7.Size of insertion: up to a few kb, specialized vectors like BACs: 100-300kb; YACs: 100- 3000kb.

7. Poly linker or multiple cloning site

8. Expression Vectors （ Expression Elements ） 1.Replicate in bacteria 2.High copies 3.Selection (antibiotic resistant gene) 4.Cloning sites 5.Promoter （ Transcription ） 6. Ribosome binding site （ Translation initiation ） 7. Termination and PolyA site 8. Some ways to control (inducible)

9. Microbial Culturing Antibiotic Selection Sterile Technique Genomic DNA Extraction DNA Precipitation DNA Quantitation GAPDH PCR Nested PCR Degenerate primers Exonuclease Gel Electrophoresis DNA Gel Interpretation Band Identification Standard Curve Use Cloning Direct PCR cloning Transformation Ligation PCR Purification Size Exclusion Chromatography PlasmidMiniprep Restriction Enzyme Digestion Gel Electrophoresis Sequencing Automated sequencing Bioinformatics Sequence Data Editing Contig Assembly Intron-Exon Prediction 10/17 10/24 10/23 Noon Colony pick 10/18 Noon Colony observation 10/12 Laboratory Overview

10. DNA Extraction Use young, fresh plant-tissue DNA extraction at room temperature Time requirement ~30 minutes Does not require DNA quantification

11. Benefits of using plants Large number of species Lots of diversity Phylogenetic （进化） approaches Avoid ethical concerns associated with animals No pre-approval

12. What are we looking for? Needs of to be a gene that is expressed in all plants: one that organisms need to maintain essential cellular functions – housekeeping gene. Examples: GAPDH Cytochrome C ATPase ß-actin

13. Why use GAPDH? Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH) 甘油醛 -3- 磷酸脱氢酶 Enzyme of glycolysis Structure and reaction mechanism well-studied Highly conserved Multitude of sequences

14. The Problem: How do we identify and detect a specific sequence in a genome? TWO BIG ISSUES: –There are a LOT of other sequences in a genome that we’re not interested in detecting. (SPECIFICITY) –The amount of DNA in samples we’re interested in is VERY small. (AMPLIFICATION)

15. The Problem: Specificity How do we identify and detect a specific sequence in a genome? Pine: 68 billion bp Corn: 5.0 billion bp Soybean: 1.1 billion bp Human: 3.4 billion bp Housefly: 900 million bp Rice: 400 million bp E. coli: 4.6 million bp HIV: 9.7 thousand bp

16. The Problem: Specificity The corn genome is 5.0 billion bp If the bases were written in standard 10-point type, on a tape measure......The tape would stretch for 7891 MILES! Identifying a 500bp sequence in a genome would be like finding a section of this tape measure only 4 feet long! How Big Is 5.0 Billion?

17. 17 The Problem: Amplification To be visible on an agarose gel, need around 10 ng DNA for fluorescent stain (or around 25ng for FastBlast). For a 500-bp product band, weighing 660 g/mol.bp, therefore need 3.03X10 -14 moles. Avogadro’s number = 6.02e23. Therefore need 1.8X10 10 copies! In other words, to “see” a single “gene”, the DNA in a sample of 100 cells would have to be multiplied 180 million times!!!!! How many molecules do we need to be able to see them?

18. 18 DNA Isolation and Amplification To identify differences in GAPDH code we must isolate plant DNA and amplify the gene of interest using PCR first with primers In some cases ， a second PCR reaction (Nested PCR) is necessary to increase specificity and yield Problems with initial PCR: –inefficient –non-specific Benefits of initial PCR: –cast a wide net –increase the pool of specific products Why is a Nested PCR reaction necessary?

19. 19 Nested PCR is more specific

20. Using Nested PCR to increase your final PCR product There is more PCR product from the nested PCR reactions since there is more specific template DNA to start from Results: intense, bold band on agarose gel DNA template: Genomic DNA DNA template: Initial PCR products Initial PCRNested PCR

21. PCR results 1% agarose gel loaded with 20 µl initial PCR samples and 5 µl nested PCR samples. ArabidopsisGreen beanLamb’s earpGAP MW ININININ 123456789 500 bp- 1000 bp- 1500 bp- 2000 bp-

22. Purification of PCR products To increase the success of ligation, it is necessary to remove unincorporated primers, nucleotides, and enzymes from the PCR reaction. Done by using size exclusion column chromatography. (In size exclusion chromatography small molecules like proteins, primers, and nucleotides, get trapped inside the chromatography beads while large molecules, like DNA fragments, are too large to enter the beads and pass through the column into the microcentrifuge tube).

23. PCR Cleaning Step-by-Step procedure 1.Resuspend the resin in the column by votexing 5 seconds 2.Remove the Cap, snap of the tip and place the column in a 2.0ml wash tube 3.Prespin the column for 0.5 minute at 3000rpm. 4.Place the column in a clean 1.5ml collection tube. 5.Apply the sample (25-100ul) to the top center of the column bed. 6.Spin the column for 1minutes at 3000 rpm 7.Save the purified sample which is in the bottom of the 1.5ml collection tube. Keep on ice. 8.Properly dispose the used column.

24. DNA Ligation

25. Different cloning procedure for PCR products

26. Pre-blunted PCR cloning vector The blunted PCR product was inserted into the vector. pJet1.2 contains a BglII restriction enzyme recognition site on either side of the insertion site. Thus, once the plasmid DNA has been isolated, a restriction digestion reaction will be performed to determine the size of the insert.

27. Blunt-End cloning of PCR products Prior to ligating the fragment into the plasmid, the PCR fragment must first be treated to remove a single adenosine nucleotide that is left on the 3′ ends of the PCR fragment by Taq DNA polymerase. This is performed by a proofreading DNA polymerase (enzymes with a 3′ proofreading exonuclease domain that allows the polymerase to remove mistakes in the DNA strands). This polymerase functions at 70oC but not at lower temperatures, so it is not necessary to inactivate this enzyme after use.

28. Ligation is the limiting step of cloning

29. 29 Procedures ：

30. 30

31. 31 Readings Molecular Cloning A laboratory manual Sambrook & Russell Cold Spring Harbor Laboratory Press c2001 Modern Genetic Analysis Griffiths, Anthony J.F.; Gelbart, William M.; Miller, Jeffrey H.; Lewontin, Richard C. New York: W. H. Freeman & Co. ; c1999W. H. Freeman & Co. Molecular Biology of the Cell Alberts, Bruce; Johnson, Alexander; Lewis, Julian; Raff, Martin; Roberts, Keith; Walter, Peter New York and London: Garland Science ; c2002Garland Science Molecular Cell Biology Lodish, Harvey; Berk, Arnold; Zipursky, S. Lawrence; Matsudaira, Paul; Baltimore, David; Darnell, James E. New York: W. H. Freeman & Co. ; c1999W. H. Freeman & Co.

32. 32 Related concepts and knowledge to discuss 1.The definition of molecular cloning. 2.How to design a primer? 3.Optimization of PCR conditions? 4.Anything eles you can think of…