Recombinant DNA Technology

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Nuclease

= hydrolyze an ester bond within a phosphodiester bond
= general term for enzymes that catalyze the hydrolysis of nucleic acid by cleaving chains of nucleotides into smaller units
-can cleave 5' or 3'

Phosphatases

= hydrolyze the ester bond in a phosphomonoester bond
= any of a number of enzymes that removes a phosphate group from a protein, reversing the action of a kinase (removes P instead of nucleotide)
-Results in 5'OH end and free P

Endonuclease

= Nucleases that cleave phosphoester bonds within a nucleic acid chain
-They may be specific for RNA or for single-stranded or double-stranded DNA
*Can be used to cleave DNA into defined fragments

Exonuclease

= Nucleases that cleave phosphoester bonds one at a time from the end of a polynucleotide chain
-They may be specific for RNA or for single-stranded or double-stranded DNA

Type II Restriction Endonucleases

= recognize short nucleotide sequences and cleave double-stranded DNA at specific sites within or adjacent to the sequences
*Same site cleavage

Recognition sequences of type II endonucleases

-Generally, but not always, 4 to 6 nucleotides in length
-Usually characterized by dyad symmetry (inverse palindromic)

Type II endonuclease cleavage

-Some cleave at the axis of symmetry, yielding "flush" or "blunt" ends.
-Others make staggered cleavages, yielding overhanging single-stranded 3′ or 5′ ends, known as cohesive termini or "sticky ends."

BamHI cleavage generates...

cohesive 5' overhanging ends

KpnI cleavage generates...

cohesive 3' overhanging ends

DraI cleavage generates...

blunt ends

What determines whether it is a 5' or 3' overhang?

The position of the cut site within the sequence
BamHI: 5' G | G-A-T-C-C 3' = 5' overhang
KpnI: 5' G-G-T-A-C | C 3' = 3' overhang

Cloning a fragment of DNA requires what?

A specially engineered vector

Recombinant DNA

A DNA molecule that has been created by joining together two or more molecules from different sources

Ligation

The process of joining together two DNA fragments
-Requires a source of energy (eg. ATP, NAD)

Cloning vector

= DNA (often derived from a plasmid or a bacteriophage genome) that can be used to propagate an incorporated DNA sequence in a host cell
-Contain selectable markers to allow identification
-Contain replication origins to maintain the vector in the host (autonomous replication)

What components are contained within a plasmid vector?

-Origin of replication
-Selectable marker gene
-Promoter
-Cloning sites (for transfer of foreign DNA)
-Leader sequences
-Tags
-Gene to be expressed

Major options to be considered when choosing a vector

-Gene dosage (copy #)
-Level of expression (determined by nature of replicon)
-Which compartment to harvest from
-Tags for purification, improvement of stability and solubility
-Codon usage (AT vs. GC rich limits availability of certain tRNAs)
-Purpose of expression: large scale industrial or analytical levels?

Replicon

= genetic unit consisting of an origin of DNA replication and its associated elements

Why is copy number important?

In case the gene has deleterious effect on the host

pBR322 Vector

-No built in promoter
-Contains the genes for resistance to ampicillin and tetracycline, with multiple cut sites within the tet resistance genes

How can pBR322 be used as a cloning vector?

Cut vector & DNA with same restriction endonuclease -> Foreign gene in

How can you determine the recombinant pBR322 molecules?

If cut site is within the tet resistance region:
1. Isolate amp E. coli cells
2. Replica plate on tet plates -> those that grew on the amp plates but not on the tet plates are recombinant molecules because the foreign gene has resulted in insertional inactivation of tet resistance

Why is selection of recombinant plasmids so important?

Because manipulations result in a variety of ligation products, including plasmids that have recircularized without insertion of new DNA (which is the most likely outcome)

pUC19 Vector

-Carries ampicillin resistance and the lacZ gene (and lac promoter)
-Multiple cloning sites within the lacZ gene

How can you determine the recombinant pUC19 molecules?

Plate cells on amp + Xgal plates:
-All are amp resistant
-Those that convert Xgal substrate to:
Blue = have functional β-gal => non-recombinants
White = nonfunctional β-gal => recombinants

How can you prevent the vector from closing in on itself?

Use 2 restriction endonucleases- use 1 to cut one end and 1 to cut the other (do the same for plasmid and introduced gene so they fit properly)

If you use 1 restriction endonucleases...

the insert can go in either orientation

If you use 2 restriction endonucleases...

the insert can go in only one orientation

How can you reduce background of a cleaved vector?

Self-ligation should be reduced by treating the EcoRI cleaved vector DNA with CIP. Although phosphatase treatment does lower the absolute efficiency of obtaining the desired DNA molecule, the great reduction in background is necessary to obtain a decent relative frequency of the desired molecule. The concentration of insert DNA(s) should be relatively high in order to facilitate ligation to the vector.

How is blunt-ended DNA joined to an EcoRI linker?

1. Use endonuclease to produce blunt ends or exonuclease to cleave off sticky ends of DNA
2. Ligate blunt-ended DNA to EcoRI linker (multiple copies of linker can be added to either end of gene)
3. Cut with EcoRI
4. Can now put in vector cut with EcoRI

What can EcoRI linker+blunt-ended DNA be used for?

To make a gene library

Primer

= a single-stranded NA molecule with a 3'OH used to initiate DNA polymerase replication of a paired template strand

Polymerase Chain Reaction (PCR)

Permits the exponential amplification of a desired sequence, using primers that anneal to the sequence of interest
*Requires sequence-specific primers

Steps of PCR

1. Denaturation (heating)
2. Annealing of primer to template (rapid cooling to primer Tm encourages primer-template hybrids)
3. Elongation (heat to optimal polymerase temp.)
-> Then, continuously repeat this cycle
-By about the 4th cycle, only the sequence b/w the primers will be amplified (=amplicon)

Why is RT-PCR useful for eukaryotes?

RNA -> cDNA -> Amplify gene
*Using RNA allows for exclusion of introns

How can PCR be used for cloning?

Put restriction enzyme sites in the primers

How do you use TOPO TA Cloning Vector to make desired product?

1a. Topo I cleaves at recognition site to produce T overhang, and Topo remains covalently linked at each end
1b. PCR product is made with A overhang to match T overhang
2. Incubate vector + PCR product at room temp.
3. Results in complementary binding, and Topos are released

How are the A overhangs produced in the PCR product?

Using Taq polymerase which leaves an A overhang

What is the disadvantage of Taq Pol? How can this be overcome?

Taq Pol has an increased error rate due to no proofreading function; Can overcome this by:
1) Can use higher fidelity polymerase, then treat with Taq for 1 cycle to produce overhang
2) Use Zero Blunt TOPO vector for blunt ends...

How do you use Zero Blunt TOPO vector to make desired product?

1a. Topo I cleaves at recognition site to produce blunt ends, and Topo remains covalently linked at each end
1b. PCR product with blunt ends is made
2. Incubate vector + PCR product at room temp.
3. Results in complementary binding, and Topos are released

PCR-Blunt II-TOPO

Contains primers for sequence analysis (M13 fwd & rvs), T7 promoter, ccdB gene, kanamycin resistance, etc.

ccdB gene

= topoisomerase poison ("natural cipro")
*Selective for insertion:
-No insert -> expresses ccdB = cell dies
-Insert -> no ccdB expression = cell viable

What are the steps of cDNA synthesis for insertion into a vector?

1. Start with mRNA with poly(A) tail
2. Prime with oligo(dT)
3. Reverse transcribe mRNA using oligo(dT) primer
4. Add RNaseH (degrades mRNA) + DNA Pol I + ligase
5. Add T4 Pol to make blunt ends
6. Methylate EcoRI cut site within gene and ligate EcoRI linkers
7. Digest linkers & size fractionate -> cDNA ready to be cloned

Why is gene methylated when preparing cDNA for vector insertion?

Methyl prevents cleavage by EcoRI in the gene of interest

How can the EcoRI-digested cDNA be fractionated?

1. Chromatography
2. Agarose gel electrophoresis

What can cDNA synthesis + vector insertion be used for?

-Make a library
-If don't know sequence

Steps for screening an cDNA expression library

1. Distribute library's clones on petri dish
2. Overlay plate with nitrocellulose paper to pick up cells
3. Lyse cells with NaOH. Proteins adhere to paper.
4. Incubate paper in solution of labeled Ab of interest (will bind protein if present)
5. Wash filter. Expose to UV light & identify fluorescent spots.
6. Compare with original plate to find bacterial clone containing human gene.

Why is screening a cDNA expression library useful?

Do not need to know the sequence of the gene

How can specific NAs be detected?

Hybridization of a labeled NA to complementary sequences can identify specific NAs
=> requires some sequence information

Probe

= a radioactive nucleic acid (DNA or RNA) used to identify a complementary fragment

Steps of hybridization to detect clones with desired DNA sequences

1. Distribute library's clones on petri dish
2. Overlay plate with nitrocellulose disk to pick up cells
3. Lyse cells and denature DNA with NaOH. Treat with UV to bind DNA to disk.
4. Add labeled cDNA probe (previously made; => have sequences info.) -> hybridizes with clone containing complementary strand
5. Wash filter. Expose to UV light (fluorescence)/ X-ray (autoradiography).
6. Compare with original plate to find bacterial clone containing the desired human genomic fragment.
-Each step requires confirmation and purification

Type of DNA Sequencing

1. Maxam Gilbert DNA sequencing
2. Sanger Method of DNA sequencing
3. Automated DNA sequencing

Maxam Gilbert DNA Sequencing

1. Remove original 5' P
2. Incubate with T4 polynucleotide kinase + γ32 P-ATP = labeled 5' end with 32P
3. Modify G with dimethylsulfide (specific for G)
4. Release modified base with heat
5. Alkali results in cleavage of phosphodiester bond, resulting in labeled fragment

What is an important requirement of Maxam Gilbert DNA sequencing?

Single-stranded DNA

What else can Maxam Gilbert DNA sequencing be used for?

Can use this method to find DNA-protein interaction=> protein will "protect" DNA from chemical reactions

Sanger Method of DNA Sequencing

= Chain termination sequencing using dideoxynucleotides (ddNTPs) to terminate DNA synthesis at particular nucleotides

Steps of Sanger Method of DNA Sequencing

1. ssDNA + 5'end primer labeled with 32P (=> all products are labeled) + DNA Pol + dATP, dGTP, dCTP, dTTP
2. Divide solution into 4 aliquots, with one type of ddNTP in each
-The actual sequence is that which is complementary to the one synthesized

ddNTPs

Lack 3'OH (instead has 3'H) => terminates synthesis because DNA Pol can no longer elongate

What is an important similarity between the Gilbert & Sanger methods?

They require ssDNA preparation

What is an important difference between the Gilbert & Sanger methods?

The Sanger method does not use harsh chemicals

How can ssDNA be prepared?

1. Phage vector
2. pBluescript
-Can initially recover in plasmid before preparation
-Contains primers for sequencing

What type of gel is used to separate DNA fragments by size?

Polyacrylamide because it can separate by as little as 1bp => better than agarose

Automated DNA Sequencing

1. Generate nested array of fragments, each with a fluorescent label corresponding to the terminating 3' base (diff. color for each base)
2. Fragments separated by electrophoresis in a single vertical gel lane
3. As migrating fragments pass through the scanning laser, they fluoresce. A fluorescent detector records the color of the passing bands, which is translated into a sequence by a computer.

When you introduce a gene to produce a protein, what do you desire?

High expression level (10-30% or more of produced protein)

What can you do if your protein of interest is toxic at high doses?

Regulate expression of the gene

What must E.coli expression vectors contain?

-E.coli expression elements
-Unique cloning sites
-An origin of replication
-A selectable marker

When you introduce a gene into E.coli, what basic elements are required?

-Repressor (to regulate gene expression)
-Promoter (-10 & -35 sequences)
-Shine Delgarno sequence (ribosome binding site)
-Terminator (stabilizes mRNA)

Why is it important to have a repressor in the introduced gene?

E.coli may negative select your introduced gene because of wasted energy for an unneeded protein => must look out for mutation

The level of regulation depends on the ____.

promoter

In what ways can promoters be induced?

Nutritional (lactose, arabinose), cold shock, physical means (pH, osmolarity), etc.

Characteristics of suitable induction sensitive promoters

-High strength
-Tight regulation - important because maintenance can be costly, gene may be lethal, etc.

IPTG

= non-hydrolyzable analog of lactose
=> no degradation and no turning off of gene due to presence of lactose

How does lactose act as an inducer?

Binds the repressor and forces it to fall off operator = derepression

Because the lac promoter is leaky, what can be done to prevent transcription?

Add glucose

What is a disadvantage of high copy # for an operon such as lac?

May lose regulation (become leaky) because not enough repressors => plasmids may be more efficient because each can carry a repressor

What are the advantages of cytoplasmic expression?

-No need for signal sequences
-High concentration of expressed protein (because spinning down cell to isolate from medium)

What are the disadvantages of cytoplasmic expression?

-Formation of inclusion bodies (unstable incorrectly folded - more common if protein made too fast/too much)
-(No disulfide bond formation)
-Protein instability

What are the advantages of periplasmic expression?

-Improved folding (no inclusion body formation)
-Disulfide bridge formation (may be enhanced by the presence of DsbA and DsbB proteins)
-Fewer proteins and possible leakage to growth medium may facilitate purification
-Less protein degradation

What are the disadvantages of periplasmic expression?

-Low protein concentration due to inefficient transport and small compartment

What is the solution to these disadvantages?

-Tight regulation of expression
-Molecular chaperones (protein specific)
-Temperature down shift after induction- slows metabolic rate, giving more time to fold properly (less formation of inclusion bodies)

Problems with expression of proteins in E. coli

Protein may be:
Unstable, toxic, insoluble, forming inclusion bodies, incorrectly folded, dependent on disulfide bridges, or active only with post-translational modifications

How can these problems be solved?

-Choice of a suitable E.coli strain
-Tags
-Fusions and leader sequences can solve many problems including disulfide bridge formation
-Proteins that need correct post-translational modifications have to be produced in eukaryotic systems

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