Cloning and Sequencing Explorer Series

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 OverviewCloning 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 Ampr 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: • 2000 to 10000bp in length; • Self replicate in bacteria • High copy numbers • Help the servive of host for selection (antibiotic resistant gene) • Multiple cloning sites • White and Blue selection (lacZ) • 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） • Replicate in bacteria • High copies • Selection (antibiotic resistant gene) • Cloning sites • Promoter （Transcription） • 6. Ribosome binding site • （Translation initiation） • 7. Termination and PolyA site • 8. Some ways to control (inducible)

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

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. Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH) 甘油醛-3-磷酸脱氢酶 Why use GAPDH? • 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 How Big Is 5.0 Billion? • 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!

17. How many molecules do we need to be able to see them? 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 1.8X1010 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!!!!!

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. Nested PCR is more specific

20. Initial PCR Nested PCR 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

21. PCR results1% agarose gel loaded with 20 µl initial PCR samples and 5 µl nested PCR samples. 1 2 3 4 5 6 7 8 9 Arabidopsis Green bean Lamb’s ear pGAP MW I N I N I N I N 2000 bp- 1500 bp- 1000 bp- 500 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 • Resuspend the resin in the column by votexing 5 seconds • Remove the Cap, snap of the tip and place the column in a 2.0mlwash tube • Prespin the column for 0.5 minute at 3000rpm. • Place the column in a clean 1.5ml collection tube. • Apply the sample (25-100ul) to the top center of the column bed. • Spin the column for 1minutes at 3000 rpm • Save the purified sample which is in the bottom of the 1.5ml collection tube. Keep on ice. • 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. Procedures：

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. ; c1999 • Molecular Biology of the Cell • Alberts, Bruce; Joh nson, Alexander; Lewis, Julian; Raff, Martin; Roberts, Keith; Walter, Peter • New York and London: Garland Science ; c2002 • Molecular Cell Biology • Lodish, Harvey; Berk, Arnold; Zipursky, S. Lawrence; Matsudaira, Paul; Baltimore, David; Darnell, James E. • New York: W. H. Freeman & Co. ; c1999

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