1. Lecture 3 Chapter 4 Molecular Cloning Methods
Molecular Biology
Lecture 3
Chapter 4
Molecular Cloning Methods
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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