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Lecture 3 Chapter 4 Molecular Cloning Methods

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1 Lecture 3 Chapter 4 Molecular Cloning Methods
Molecular Biology Lecture 3 Chapter 4 Molecular Cloning Methods Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

2 Lecture outline Gene cloning Restriction endonucleases Plasmids pBR322
pUC Selection

3 Gene Cloning Introduce a foreign gene or piece of DNA into a suitable vector and inserting the recombinant molecule into a bacterial host Cloning can also be done in eukaryotic cells such as yeast One can then produce large quantities of the gene or piece of DNA in pure form

4 The Role of Restriction Endonucleases
Restriction endonucleases, first discovered in the late 1960s, are named for preventing invasion by foreign DNA by cutting it into pieces These enzymes cut at sites within the foreign DNA instead of chewing from the ends By cutting DNA at specific sites they function as finely honed molecular knives

5 Restriction-Modification System
What prevents these enzymes from cutting up the host DNA? They are paired with methylases Theses enzymes recognize, and methylate the same site The sequence specific methylase and restriction endonuclease are called a restriction-modification system, R-M system Methylation protects DNA, after replication (the parental strand is already methylated)

6 Restriction Endonuclease Specificity
A 6-bp cutter will yield DNA fragments averaging 4000-bp or 4 kilobases (4kb) in length e.g. EcoR1 recognize the sequence GAATTC CTTAAG Probability of finding the sequence 1/.25 X .25 X .25 X .25 X .25 X .25 = 4096

7 Restriction Endonuclease Specificity
Restriction endonucleases recognize a specific DNA sequence, cutting ONLY at that sequence These enzymes can recognize 4-bp, 6-bp, 8-bp sequences The frequency of cuts lessens when the recognition sequence is longer

8 Use of Restriction Endonucleases
Many restriction endonucleases make staggered cuts in the 2 DNA strands This leaves single-stranded overhangs, called sticky ends that can base-pair together briefly This makes joining 2 different DNA molecules together much easier Staggered cuts occur when the recognition sequence usually displays twofold symmetry, palindromes

9 Use of Restriction Endonucleases
Example EcoR1 This enzyme leaves single-stranded overhangs, called sticky ends that can base-pair together GAATTC G AATTC CTTAAG CTTAA G + EcoR1 = and

10 Use of Restriction Endonucleases
Making a recombinant DNA molecule in presence of DNA ligase and ATP + GAATTC CTTAAG DNA ligase will synthesize the phosphodiester bonds

11 Summary Restriction endonucleases recognize specific sequences in DNA molecules and make cuts in both strands This allows very specific cutting of DNAs The cuts in the two strands are frequently staggered, so restriction enzymes can create sticky ends that help to link together 2 DNAs to form a recombinant DNA in vitro

12 Vectors Vectors function as DNA carriers to allow replication of recombinant DNAs Typical experiment uses 1 vector plus a piece of foreign DNA Foreign DNA has no origin of replication, the site where DNA replication begins Depends on the vector for its replication There are 2 major classes of vectors: Plasmids Phages (not covered in this course)

13 First cloning Experiment Using Restriction Endonuclease
An early experiment used EcoRI to cut 2 plasmids Small circular pieces of DNA independent of the host chromosome Each plasmid had 1 site for EcoRI Cutting converted circular plasmids into linear DNA with the same sticky ends The ends base pair Some ends re-close Others join the 2 pieces DNA ligase joins 2 pieces with covalent bonds

14 Plasmids As Vectors pBR plasmids were developed early but are rarely used today pUC series is similar to pBR 40% of the DNA, including tetracycline resistance has been deleted Cloning sites are clustered together into one area called the multiple cloning site (MCS)

15 pBR322 Plasmid pBR322 illustrates cloning methods simply
Resistance for 2 antibiotics Tetracycline Ampicillin Origin of replication between the 2 resistance genes Only 1 site for several restriction enzymes

16 pBR322 Cloning Clone a foreign DNA into the PstI site of pBR322
Cut the vector to generate the sticky ends Cut foreign DNA with PstI also – compatible ends Combine vector and foreign DNA with DNA ligase to seal sticky ends Now transform the plasmid into E. coli

17 Bacterial Transformation
Traditional method involves incubating bacterial cells in concentrated calcium salt solution The solution makes the cell membrane leaky, permeable to the plasmid DNA Newer method uses high voltage to drive the DNA into the cells in process called electroporation

18 Screening Transformants
Transformation produces bacteria with: Religated plasmid Religated insert Recombinants Identify the recombinants using the antibiotic resistance Grow cells with tetracycline so only cells with plasmid grow, not foreign DNA only Next, grow copies of the original colonies with ampicillin which kills cells with plasmid including foreign DNA

19 Screening With Replica Plating
Replica plating transfers clone copies from original tetracycline plate to a plate containing ampicillin A sterile velvet transfer tool can be used to transfer copies of the original colonies Desired colonies are those that do NOT grow on the new ampicillin plate

20 Directional Cloning Cut a plasmid with 2 restriction enzymes
Clone in a piece of foreign DNA with 1 sticky end recognizing each enzyme The insert DNA is placed into the vector in only 1 orientation Vector religation is also prevented as the two restriction sites are incompatible

21 pUC and b-galactosidase
Newer pUC plasmid

22 pUC and b-galactosidase
Newer pUC plasmid lac promoter O MCS lacZ (a-peptide) Induced by lactose or IPTG Under the control of the lac repressor The MCS is a cluster of sequences recognized by restriction endonucleases

23 pUC and b-galactosidase
Clones with foreign DNA in the MCS disrupt the ability of the cells to make b-galactosidase b-galactosidase is encoded by the lacZ gene of the lac operon b-galactosidase cleaves lactose and can also cleave the synthetic substrate X-gal. Cleaved X-gal gives a blue coloration. You can monitor the activity of b-galactosidase by looking at the blue coloration

24 pUC and b-galactosidase
a-complementation Plasmid contains part of the lacZ gene coding for the N-terminal extremity of the b-galactosidase enzyme. When expressed in E. coli lacZ- strain = no activity Host bacterial strain contains a truncated lacZ gene encoding a polypeptide missing the N-terminal extremity When expressed in E. coli = no activity

25 pUC and b-galactosidase
a-complementation When the plasmid is introduced in the bacterial strain containing the truncated enzyme, activity is recovered. The two partial gene products can cooperate to form an active enzyme (model on the blackboard)

26 Summary First generation plasmid cloning vectors include pBR322 and the pUC plasmids pBR322 has 2 antibiotic resistance genes Variety of unique restriction sites for inserting foreign DNA Most of these sites interrupt antibiotic resistance, making screening straightforward (but requires replica plating) pUC has Ampicillin resistance gene MCS that interrupts a b-galactosidase gene MCS facilitates directional cloning into 2 different restriction sites


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