MICR5831 L6: DNA Cloning I 7/29/25

Describe the two approaches to cloning genes (slide 8)

1) Random Cloning:

-Cloning random restricted DNA fragments

-Use restriction enzymes

-Do not need to know sequence of target gene

-Not specific for gene of interest (need phenotype)

-Wrong RE can cut inside gene of interest

2) PCR Cloning:

-Cloning specific PCR fragments

-Very specific amplification of gene

-Must know sequence of target gene for design of primers

What are the methods needed for cloning (slide 9)

1) Method for isolating genomic DNA from donor

2) Method for fragmenting donor and recipient vector DNA

3) Method of ligating the two pieces of DNA together

4) Method for transferring the recombinant molecule → E. coli host

5) Method for identifying which bacterial transformants carrying the recombinant plasmid of interest

6) Methods for purifying and analyzing recombinant plasmid

What are the properties of restriction enzymes (slide 10)

Type I:

-Cleave the phosphodiester bond at specific sites in the DNA duplex (restriction site)

-Requires Magnesium for activity

-Restriction site is 4-10 bp palindrome

Type II: (Important)

-Cuts fixed position, sticky or cohesive ends, blunt ends

-Used in cloning experiments

Type I and Type III: More irrelevant

-Cuts random site outside recognition sequence

-Not used in cloning experiments

What is a palindrome in DNA and what is the role of this in restriction digestion? (slide 10 and 13)

-Sequence of one strand reads in reverse order to that of the complementary strand

-Restriction site is a palindrome

-Role: Provide recognition sites to restriction enzymes

Using a figure describe "sticky" and "blunt" ended DNA (slide 11 and 12)

Sticky Ends:

-EcoRI (5' overhang)

-PstI (3' overhang)

Blunt Ends:

-SmaI

What is an Isoschizomers? (slide 12)

-REs that recognize the same palindrome

-Produce either same or different digestion products

How is the frequency of cutting by restriction endonucleases determined (slide 14)

-Related to length of recognition sequence

-Probability that each bp is found consecutively

-1 site per 256 bp (1/4^4) for a 4 base cutter recognizing one sequence

-Must take into account specificity of RE (HincII cuts 1/1024 bp)

What are the properties of agarose gels (slide 14)

-Polymer of repeating di-galactan unit

-Tris-acetate-EDTA (TAE)/Tris- borate-EDTA (TBE) buffer pH8

-Heating agarose -> soluble -> cast gel

-Cooled -> porous gel

-Concentration determines porosity

-Large fragment migrates slower than small

How does an agarose gel separate DNA fragments? (slide 15)

-DNA molecules separated according to length during migration through porous gel

-DNA negatively charged at pH 8

-DNA migrates towards positive electrode when subjected to an electric current

-Visualization: Ethidium bromide stain (cancer), UV fluorescence

What are the features and properties of cloning vectors (slide 18)

-Small plasmids

-Selectable marker (antibiotic resistance)

-Multiple restriction enzyme sites for cloning

-Origin of replication (oriV, low copy 1-2 high copy >50)

-Mechanism for detecting recombinant vectors: blue white screening, antibiotic resistance gene as marker

What are the components in a DNA ligation reaction? (slide 19)

-DNA with compatible ends

-Molar ratio of vector to insert is 1:3

-T4 DNA ligase from T4 bacteriophage

-100x more enzyme for blunt ends

-ATP

-Mg++

-Optimal Buffer pH

-Incubation at 16C

How can the re-ligation of vector be prevented? (slide 20)

Alkaline Phosphatase (ALP):

-Removes 5' phosphates from the termini of cut DNA

-Must be removed/ destroyed before ligation

Sources of AP:

-Bacterial

-Calf intestinal

-Shrimp

How can cleaved DNA be treated to create compatible ends? (slide 21)

1) Klenow fragment of DNA polymerase I:

-Fill in 5' overhangs by 5'→3' DNA polymerase activity

-3' → 5' exonuclease activity removes 3' overhangs (sticky ends)

2) T4 DNA polymerase

-Same activities as Klenow

-Exonuclease activity 200x that of Klenow

Describe the chemical method for bacterial transformation (slide 24)

-Log phase of E. coli culture

-Centrifuge

-Resuspend bacterial pellet in CaCl2 solution

-Chill on ice

-Aliquot competent cells, store at 80C and amplify plasmid DNA then chill on ice

-Heat shock in 42C water bath

-Plate on LB and ampicillin

-10E6-10E8 colonies/ug DNA

What is this?

-Plasmids

-Circular (single or multiple copies)

-Supercoiled dsDNA (2-200 kb)

-Replicate independently of the host chromosome

True or False: Genes carried on plasmids are essential to the host's survival

False, plasmid genes may be useful in certain environments but will move in/out of bacteria

True or False: Since Plasmids don't belong to the genome, they can be spit out if unhelpful

True

What are some genes that might be carried on a plasmid?

-F-plasmid

-Resistances e.g. antibiotics, pesticides

-Virulence genes

-Metabolism

What is an example of a bacteria with a plasmid that confers huge genetic islands of resistance?

Staphylococcus aureus

What is this?

-Restriction Endonucleases (RE)

-Enzymes that can cleave the phosphodiester bond at restriction sites

-Restriction site is a 4 -10 base pair palindrome

-Mg2+ needed for activity

What is needed for RE activity?

Magnesium (Mg2+)

What is EcoRI named after?

Escherichia coli strain R

What is this?

-Restriction site

-Specific sites in the DNA duplex

-Cleaves the phosphodiester bonds here

-4-10 base pair palindrome

-Sequence of one strand reads in reverse order to that of the complementary strand

What Type of RE is this?

-Cuts at fixed position within restriction site

-Sticky or cohesive ends

-Blunt ends

-Used in cloning experiments

Type II Restriction Endonuclease

What Type of RE is this?

-Cut at random sites outside recognition sequence

-Binds to restriction sequence and cuts bp up or downstream

-Not used in cloning experiments

Type I and Type III Restriction Endonucleases

What ends do these RE produce?

-AluI

-HaeIII

Hint: Alucard is in Hell for using blunt force trauma.

Blunt ends

What ends do these RE produce?

-BamHI

-EcoRI

-HindIII

Sticky ends

What is this?

-Isoschizomers

-Recognise the same palindrome and produce either different or the same digestion products

-Can be uses to directionally clone

What is this?

-Sticky Ends

-Cuts at beginning or end of recognition sequence

-Phosphate/hydroxyl at end of cuts, used to stick DNA back together

-5' or 3' overhang

What is this?

-Blunt ends

-Cuts right in middle

-Always even number of nucleotides

-Nondirectional cloning

True or False: Some Type II REs recognise more than one palindrome

True

Why are some Type II REs able to recognize more than one palindrome?

-Allow substitutions in one or more positions of the recognition sequence

What is an example of a Type II RE that can recognize more than one palindrome?

-HincII

-Has substitutions in the two central positions of the recognition sequence

-But not at the top or bottom

How are purines (A, G) designated?

Memory: Pure silver

R

How are pyrimidines (C or T) designated?

Memory: See the pyramids, sip some Tea

Y

What ends do these have?

-EcoRI

-HindIII

-XmaI

Sticky (5') ends

What ends do these have?

-EcoRV

-HincII

-SmaI

Blunt ends

What ends do these have?

-KpnI

Sticky (3') ends

What determines the frequency of the RE cuts?

Length of the recognition sequence

If a 4 base cutter recognizes 1 sequence, how many bases per site will the fragments be?

4-mer = (1/4)^4 = 1/256

1 site per 256 bases

How many bp does HincII cut?

1/1024 bp

If a 6 base cutter recognizes 1 sequence, how many bases per site will the fragments be?

(1/4)^6 = 1/4096

1 site per 4096 bp

What is this?

-Agarose

-Polymer of repeating di-galactan unit

-Separates DNA molecules according to length during migration through gel

-Porosity determines concentration

Which DNA fragment will move slower through agarose gel, a large or smaller one?

Larger

Which separates DNA fragments better, higher concentrated gel or lower concentration gel?

Higher concentration

How do you produce cast gel out of agarose?

-Heat it up

-Gel becomes soluble

How do you produce soluble gel out of agarose?

Cool it down

What repeating unit does agarose gel have?

Di-galactan

What buffers does agarose gel have?

-Tris-acetate-EDTA (TAE)

-Tris-borate-EDTA (TBE)

-pH of 8

What happens when DNA is at pH 8 and subjected to an electric current?

Negatively-charged DNA migrates towards positive electrode

What can you use to visualize DNA during agarose gel electrophoresis?

-Ethidium bromide (carcinogen)

-UV fluorescence

-Nontoxic dye: Cytogreen

What is this?

-DNA Cloning

-Isolates genes from the rest of the genome

-Amplifies DNA, makes multiple copies

-Good for DNA mapping, sequencing, unknown gene identification

How does DNA cloning allow for DNA manipulation?

-Study mutation gene function and regulation

-Express and purify proteins to study structure

How do you create a recombinant vector?

-Use restriction enzyme to cut vector plasmid up and digest it

-Take cut vector, insert new genes

-Ligate genes to cut vector

What is this?

-Hemolysin

-Enzyme that lyses/disrupts RBCS membrane

-Gene (cleR) -> Protein (CleR)

-Carried by Streptococcus

How can you detect the presence of hemolysin?

-Use blood agar

-Zone of hemolysis around bacteria colonies

What are some possible strategies to move cleR gene from source DNA into a recipient vector?

-Random cloning of fragmented genomic DNA via common restriction site

-Specific cloning of PCR-amplified DNA

Name an example of a destination for cleR

Cloning vectors/plasmids

Name an example of a destination host for cleR

-E. coli K12 usually

-Yeast host also possible, but other hosts rare

If the DNA sequence of cleR is unknown and there is no prior work, how would you isolate the gene of interest?

-Search for gene with same phenotype only

-Identification via sequence similarity to other known hemolysins

What are the pros of cloning randomly restricted fragments via restriction enzymes?

Do not need to know the sequence of the target gene

What are the cons of cloning randomly restricted fragments via restriction enzymes?

-Not specific for gene of interest

-RE may cut inside gene of interest, so need to try different RE

What are the pros of PCR cloning?

Very specific amplification of gene

What are the cons of PCR cloning?

Must know sequence of target gene for design of primers

Name this method for isolating genomic DNA from donor Streptococcus:

-High quality, high Molecular Weight DNA

Random cloning

Name this method for isolating genomic DNA from donor Streptococcus:

-Low quality DNA (boil prep) sufficient for PCR

-Just emulsify in water

PCR

Name this enzyme used for fragmenting/cutting donor DNA and recipient vector DNA

-Restriction Endonucleases/Enzyme

-Need to know restriction sites

Name this enzyme used for ligating the two pieces of DNA together

T4 DNA ligase

Name this method used for transferring the recombinant molecule into E. coli so it can replicate

Bacterial transformation

What is this?

-Small plasmids

-Selectable marker for antibiotic resistance

Cloning vectors

True or False: Cloning vectors only have one restriction enzyme site

False, cloning vectors can have multiple cloning sites

What is this?

-Cloning vector site where plasmid starts replication

-Determines if low copy or high copy

-Low copy: Stringent 1-2 copies

-High copy: Relaxed >50 copies

Origin of replication (oriV)

How do you detect recombinant vectors?

-Insertional inactivation

-Blue-white screening

-Antibiotic resistance gene as marker

What is this?

-Phosphodiester bond between the 3' hydroxyl of one nucleotide and the 5' phosphate of another

Ligation

Ligation is a phosphodiester bond between a 3' (Blank 1) and 5' (Blank 2)

1) Hydroxyl (3')

2) Phosphate (5')

What are the requirements for a successful DNA ligation?

1) DNA with compatible ends

2) T4 DNA ligase from T4 bacteriophage

3) ATP

4) Mg2+

5) pH optimal buffer

6) Incubation at 16C

What is the molar ratio of Vector to Insert in DNA ligation?

1:3

How can you stop your vector from self-ligating, causing complementary sticky ends to get stuck together?

Alkaline Phosphatase (AP)

What is this?

-Alkaline Phosphatase

-Removes 5' phosphates from cut DNA terminus

-Must be removed/destroyed before ligation

What are some sources of Alkaline Phosphatase, the enzyme that removes 5' phosphates from cut DNA termini?

-Bacteria

-Calf intestine

-Shrimp

When should you remove/destroy Alkaline Phosphatase?

Before ligation occurs

What are compatible ends?

-All blunt ends

-Cohesive ends that are identical

What can you use to make all cohesive ends into blunt ends so they will become compatible ends?

1) Klenow fragment of DNApol I

2) T4 DNA Polymerase

What is this?

-Helps create blunt ends that are compatible

-Fill in 5' overhangs

-5'→3' DNA polymerase activity

-3' → 5' exonuclease activity removes 3' overhangs

Klenow fragment of DNA polymerase I

What is this?

-Helps create blunt ends that are compatible

-Fill in 5' overhangs

-5'→3' DNA polymerase activity

-Exonuclease activity is 200x stronger

T4 DNA Polymerase

If the Klenow fragment of DNAPol I and T4 DNAPol are responsible for filling in 5' overhangs:

-What thermostable DNAPol is responsible for producing 3' overhangs?

Taq (Thermus aquaticus)

What is this?

-Cloning process where Taq adds 3' overhangs to PCR product

TA Cloning

Before cloning into blunt restriction site via Taq (Thermus aquaticus), what should PCR fragments be treated with?

Klenow/T4 DNAPol

What are some thermostable enzymes used in PCR that also produce blunt-ended DNA fragments via 3'->5' exonuclease proofreading?

-Pfu (Pyrococcus furiosus)

-Kod (Thermococcus kodakaraensis)

What is a disadvantage of TA cloning (using Taq to add 3' overhangs to PCR fragments)?

-Nondirectional cloning, can go either way

-Low efficiency using blunt ends instead of sticky

-Limited to Taq fragments

What are some benefits of incorporating Restriction sites into PCR primers at the 5' end?

-Sticky end ligation is efficient

-Directional cloning when 2 different restriction sites are incorporated

Name the steps for expressing a cloned gene in a new host

1) DNA ligation of cleR into new vector

2) Transformation into E. coli and plasmid replication

3) Bacteria plated on Medium + Antibiotic

4) Vector containing insert survives, forms clones

5) Clones incubated overnight

6) Blood Agar plate inoculation

7) Hemolysis observed, confirms gene expression

What type of Transformation is used here to express a clone gene in a new bacterial host?

1) Log phase of E. coli culture, centrifuge

2) Resuspend bacterial pellet in CaCl2 solution

3) Chill on ice

4) Aliquot competent cells, store at 80C while plasmid DNA gets slowly amplified

5) Heat shock cells

6) Plate on LB Medium + Ampicillin

7) 10E6-10E8 colonies/ug DNA that are ampicillin resistant

Chemical Method: Cloning small fragments, <20 kB

What type of Transformation is used here to express a clone gene in a new bacterial host?

1) Log phase of E. coli culture, centrifuge

2) Resuspend bacterial pellet in sterile H₂O

3) Chill on ice, centrifuge and resuspend bacterial pellet in sterile H2O

4) Amplify plasmid DNA and use competent cells immediately

5) Electroshock

6) Add to microtube with saline buffer

7) Place on LB Medium + Ampicillin

8) 10E8-10E10 colonies/ug DNA that are ampicillin resistant

Electroporation: Cloning fragments >20kB