Lecture 20: Recombinant DNA cloning

Define Recombinant DNA

Any DNA molecule formed by joining DNA segments from different sources

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Restriction endonucleases - enzymes

cleave DNA at specific internal sequences (digestion)

Agarose

* large DNA fragments (>500 nucleotides)
* moderate resolution (100s of nucleotides)
* horizontal gel
* standard visualization (EtBr, radiography, etc)
* pulsed-field (charge electric field) - extremely large fragments

Polyacrylamide gel electrophoresis (PAGE)

* Small DNA fragments (

What are the contributing factors for the specificity of DNA annealing?

* strand length (longer strand = more stable)
* pH, salt, temp ( extreme pH, high salt, or high temp = destabilizing)
* sequence (G-C stronger than A-T, mismatches reduce Tm)
* can be DNA/DNA, DNA/RNA, RNA/RNA
* use of fluorescent dsDNA binding molecule can “report” the proportion of dsDNA

i**ncrease/decreases** stringency and **increase/decrease** sensitivity = good hybridization

increase stringency and increase sensitivity

explain PCR

allows amplification of any gene via base pairing specificity

Elements of operons

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* regulatory gene
* operator
* promoter
* structural gene

regulatory gene

codes for a protein that binds DNA and influences transcription of the structural genes (usually a repressor)

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Operator

DNA sequences that binds the regulatory protein

Promoter

DNA sequence that binds RNA polymerase in absence of repressor

structural gene

codes for non-regulatory proteins(s) that have activity in metabolism, etc

The lac operon is composed of what 4 genes?

* lacZ
* lacY
* LacA
* lacL

Lac Z

* beta-galactosidase


* Cleaves lactose -→ galactose and glucose

allolactose

minor product of lactose hydrolysis by beta-galactosidase and is the **natural inducer** that bind to lac repressor.

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Steps for Cloning Genes for expression.

1. plasmid is cut with 2 different restriction enzymes
2. gene is amplified by PCR from genomic DNA
3. primers contain same restriction sites as used to cut plasmid
4. gene is cut to give sticky ends that are complementary to plasmid
5. cut gene is ligated to plasmid by DNA ligase
6. plasmid with cloned gene is added to E.coli in presence of antibiotic

_____ is a common host for high-level expression of proteins

E.coli

Advantages of using recombinant DNA for drug development? (4)

1. source availability
2. safety
3. engineering
4. economical

Recombinant DNA processes

* DNA digestion and ligation
* gel electrophoresis
* hybridization
* PCR
* transformation of host cells

sticky ends

single stranded ends of DNA after a cut

Explain the process of Cutting and pasting DNA

* two DNA pieces with the same unique restriction site on each end can be digested with restriction enzymes
* mixed and treated with DNA ligase to give joined fragment

what enzyme is used for cutting and pasting DNA?

Eco RI restriction enzyme DNA ligase

restriction enzymes

each gives unique pattern for a given DNA substrate

what happens when gene inserted after lac promoter?

* it can be induced by addition of IPTG
* large amounts of protein can be made in E.Coli

Silica based DEAE resin

positively charged at pH 7 and bidns DNA with high capacity

DNA annealing

* high temp or high pH causes denaturation to single strands
* slowly cool or lower pH causes restoration of DNA double helices

Define Tm

temp at 50% of DNA annealed (50% double-stranded)

Modulating DNA annealing specificity

with appropriate stringency and sensitivity, hybridization based techniques can detect complementary sequences present at the concentration of one molecule per cell

Example of increasing sensitivity

single stranded DNA probes only for gene A

Lac Y

galactoside permease required for transporting lactose across membrane

Lac A

transacetylase detoxifies other permease substrates by acetylation

regulatory gene of lac operon

LacL

Lac L

encodes lac repressor (binds to operator sequence and can block transcription)

allolactose binding to repressor

binding prevents repressor binding and allowing transcription of structural genes

basic bacterial expression plasmid componenets

* poly linker
* origin of replciation
* ampicillin reisstance
* lac repressor

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polylinker

allows choice of variety of restriction sites, aslo called multicloning sites

origin of replciation

dictates number of plasmid copies per cell

antibiotic resistance gene

allows constant selection for bacteria that are replciating the plasmid

polyhistidine fusion tag

can be added to the N-terminus of your protein so it can be easily purified by metal ion chelation

in the presence of IPTG

\-lac promoter in genomic DNA is free \n -Ec RNA polymerase transcribes T7 RNA polymerase gene, which synthesizes large amounts of mRNA for your gene only \n -T7 promoter only found in plasmid

Taq polymerase

lacks a proofreading exonuclease and is more error prone

Proofreading thermostable polymerase

* Used for increases fidelity

What are used as PCR templates?

* DNA or RNA

PCR primers

Can contain labels, engineered restriction sites, biotin

PCR cycles

Runs for 30 cycles

2-10 mins each cycle

Structural genes of lac operon

* lacZ
* lacY
* LacA

beta galactosidase production

produced in small amounts in absence of lactose, however in presence of lactose, the number of enzyme molecules rise dramatically

Operon

group of genes whose expression is corregualted in response to precne of absence of lactose as carbon source

Inducer in Lac operon

Allolactose, binds repressor and prevents it form binding operator DNA sequence

RBS and start codons

In operons, usually much closer to the stop codon of the upstream gene