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Cloning and Sequencing Explorer Series

鲁林荣

娄 军方 瑜

Related knowledge

• Molecular cloning

• Plasmid/vector

• DNA sequencing technology

• DNA sequence analysis

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.

Plasmid

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

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.

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.

Poly linker or multiple cloning site

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)

Microbial CulturingAntibiotic SelectionSterile Technique

Genomic DNA ExtractionDNA PrecipitationDNA Quantitation

GAPDH PCRNested PCRDegenerate primersExonuclease

Gel ElectrophoresisDNA Gel InterpretationBand IdentificationStandard Curve Use

CloningDirect PCR cloning

Transformation

Ligation

PCR PurificationSize Exclusion Chromatography

Plasmid MiniprepRestriction Enzyme DigestionGel Electrophoresis

SequencingAutomated sequencing

BioinformaticsSequence Data EditingContig AssemblyIntron-Exon Prediction

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10/23 Noon Colony pick

10/18 Noon Colony observation

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Laboratory Overview

DNA Extraction

• Use young, fresh plant-tissue

• DNA extraction at room temperature

• Time requirement ~30 minutes

• Does not require DNA quantification

Benefits of using plants

• Large number of species

• Lots of diversity

• Phylogenetic （进化） approaches

• Avoid ethical concerns associated with animals

• No pre-approval

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

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

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)

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

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?

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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!!!!!

How many molecules do we need to be able to see them?

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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?

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Nested PCR is more specific

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 PCR Nested PCR

PCR results

1% agarose gel loaded with 20 µl initial PCR samples and 5 µl nested PCR samples.

Arabidopsis Green bean Lamb’s ear pGAPMW

I N I N I N I N

1 2 3 4 5 6 7 8 9

500 bp-

1000 bp-

1500 bp-2000 bp-

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 sizeexclusion chromatography small molecules like proteins, primers, andnucleotides, get trapped inside the chromatography beads while largemolecules, like DNA fragments, are too large to enter the beads and passthrough the column into the microcentrifuge tube).

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.

DNA Ligation

Different cloning procedure for PCR products

Pre-blunted PCR cloning vector

The blunted PCR product was insertedinto the vector. pJet1.2 contains a BglIIrestriction enzyme recognition site oneither side of the insertion site. Thus,once the plasmid DNA has beenisolated, a restriction digestion reactionwill be performed to determine the sizeof the insert.

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.

Ligation is the limiting step of cloning

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Procedures ：

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Readings • Molecular Cloning A laboratory manualSambrook & 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, PeterNew 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

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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…