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RECOMBINANT DNA TECHNOLOGY

Recombinant DNA – combination of DNA from 2 different sources

Restriction Enzymes (Endonucleases) and Restriction Fragments

Enzymes which recognize a specific nucleotide sequence (4-8 bp, usually palindromic), cuts at defined sites

3 types; but type II RE is most frequently used

Obtained from microorganisms, nomenclature based on source (Gsp [Genus, species] strain number); generates 2 kinds of ends:

o Sticky / cohesive ends – due to asymmetrical cleavage, produces overhangs and recessed ends; eg. EcoRI

Base-pairing is possible if sticky ends are complementary (cut by same RE); covalently linked by DNA ligaseo Blunt / flushed ends – symmetrical cleavage; joined by T4 ligase; eg. SmaI

Separation of DNA fragments via Gel Electrophoresis

Separates based on size; smaller fragments move faster and farther 

Polyacrylamide gel – separates DNA (1000 bp), high resolving power, differentiate upto 1 nucleotide, determine base sequences

Agarose gel – for larger DNA (20 kbp), easier, horizontal

DNA ladder – used along with specimen, known MW DNA fragments for comparison

Visualization via autoradiography (radioactively labeled DNA) or fluorescence (ethidium bromide stain)

Southern Blotting 

By EM Southern, detects DNA fragments with specific base sequences

Electrophoresis in agarose gel

fragments denatured into ssDNA with alkali and heat

Gel on sponge over buffer; covered by nitrocellulose (NC) paper  buffer flow through gel transfer of fragments to NC = replica

Addition of probe (32P ssDNA) (80°C, overnight) hybridize with specific complementary fragment wash to remove unbound

probes autoradiography on x-ray or fluorescence = exposed portions = molecules complementary with probe

Northern blot = for RNA, uses nucleotide probe; more difficult because RNA is unstable

Western blot = for protein, uses antibody probe

DNA Sequence Analysis

Maxam-Gilbert Method / Chemical method

o DNA fragment labeled with 32P at one end divided into 4 samples

o Each sample + chemical that specifically destroys 1 or 2 nucleotide base (destroys G, A/G, T/C, and C); few sites

nicked

o

Samples run on polyacrylamide gel

autoradiography

read (bottom-up) Sanger Method / Enzymatic / Dideoxy/Chain termination method

o DNA denatured into ssDNA (complementary strand will be made) divided into 4 samples

o32P at 5’ at 3’ end of template ssDNA + vector (for introduction into host cell) each sample gets all dNTP + 1 of each

ddNTPo Chain elongation with DNA polymerase ends at ddNTP (cannot form phoshodiester bond)

o Samples run on polyacrylamide gel autoradiography read (bottom-up) results will show cDNA sequence must

convert to template strand via base-pairing

Construction of Recombinant DNA and Cloning 

Cloning vectors – delivers DNA into host cell

o Plasmid – dsDNA, closed circular, extrachromosomal, 1-200 kbp, replicates independently, has:

antibiotic resistance genes (selectable markers), restriction sites (cloning sites), origin of replication (copy

# control)

o Bacteriophage lambda – dsDNA, linear, for eukaryotic cloning

o Cosmids – plasmids with bacteriophage DNA sequence

o ss bactriophage vectors – for DNA sequencing; M13

o Expression vectors – has very strong promoter regions

Steps (in-vivo cloning – cell based, requires vectors)

o Foreign DNA & vector (has antibiotic resistance) cleaved by same RE combined with DNA ligase = recombinant DNA

o Transformation (prokaryotes) / Transfection (eukaryotes) – introduction of rDNA into host via:

CaCl + heat shock – most common, 20 min, 40°C

CaPO4-mediated transfection

Protoplast infusion by polyethylene glycol (PEG)

Microinjection – inject DNA directly into cello Allow growth in colonies in medium with antibiotics

o Screening via:

Antibiotic resistant colonies – Ampicillin – Tetracyclin; *insertional inactivation

Gal-X – presence stains colonies blue

Identification of individual phage plaques

Colony hybridization technique

• Colonies blotted in NE denaturated with alkali radioactive probe hybridize with NE

autoradiography match colored and original colonies

o Library – selected colonies grown in large cultures complete set of clones in host cell

Genomic – all genomic DNA (exons + introns)

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cDNA – expressible DNA only (exons)

o Obtaining DNA from library: cell lysis plasmid cut with RE fragments run in agarose gel

cDNA Cloning

o from mRNA, uses reverse transcriptase (AMV, MLV), produces dsDNA

o dsDNA ligated inserted into vectors (similar to genomic cloning) = rDNA host cell transformation / transfection

colony propagation selected, screened cDNA library

o cDNA ligated into eukaryotic expression vector  protein expression, isolation, purification

Thermocyclers (Mullis) / Polymerase Chain Reaction

o

In-vitro, rapid, mass production of DNA from small pieces, amplifies DNA, for clinical / forensicso Requirements: DNA, heat stable DNA polymerase (Taq), all 4 dNTPs, 2 oligonucleotide primers complementary to base

sequence in each DNA strand, flanks region to be amplified

o Steps: Mix  Heat to 94°C for denaturation  Cool for annealing and extension  repeat 20-30 times

 Applications

Protein Mass Production – in E.coli

Medical Diagnosis – cancer detection, detection of polymorphisms via RFLP (DNA fingerprinting), testing for mutations via PCR

Gene Therapy – cure genetic diseases by introducing normal genes, currently experimental

Genetic Counseling – test for possibility of genetic disease, for decision-making in matrimony or child-bearing

Construction of Useful Organisms – bacteria that can metabolize petroleum, bacteria that can produce chemicals, GMOs