Recombinant DNA Technology

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Recombinant DNA Technology

Dates in the Development of Gene Cloning:

1965 - plasmids 1967 - ligase

1970 - restriction endonucleases

1972 - first experiments in gene splicing 1974 - worldwide moratorium declared

1975 - Asilomar Conference to determine how to proceed 1979 - guidelines relaxed

Overview of Basic Cloning Experiment

Properties of Vectors for Transferring Genes 1. able to enter host

2. replicate in host (ori for DNA replication)

3. selectable marker genes to identify it, ie. sugar metabolism, antibiotic resistance

Types of Vectors:

1. Phages (λ or M13)

lambda (λ) - accept large DNA fragments (40 kb)

M13 - (10 KB fragments) unique promoters that aid in DNA sequencing.

2. Plasmids (pBR322) - ideal vectors to carry foreign DNA transferred to host cell -small, antibiotic-resistance genes

-often contain polylinkers - multiple unique RE sites -ORI

-some have a eukaryotic transcriptional promoter

3. YAC – yeast artificial chromosome, ORI, centromere for segregation, telomeres for stability

Shuttle vectors – used in more than one type of host, plasmids that can move between prokaryotic and eukaryotic cells, ex. must have marker genes for E. coli and S.

cerevisiae

- eukaryotic transcriptional promoter in prokaryotic vector Expression vectors –

- examine the regulation of a gene (how initiation of transcription is controlled by promoter)

How - promoter and translational start sequence placed next to insertion site, connect the promoter to a reporter gene (GFP is used) - gene that can be easily assayed

-use inducible promoter Restriction Endonucleases

1. restriction - modification system; defense against foreign DNA

- methylation of RE target sequences

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2. several classes - Type II that cut in target sequence; recognize palindromes 3. Types of cuts

a. cohesive or sticky ends - reanneal with ligase b. blunt ends - can be ligated with T4 ligase 4. Other considerations

type of cut, cleavage site, parameters for the reaction to occur 5. Typical number of RE cuts:

- bacteria 2 x 10

⁶

bp - 100-1000 fragments - euk cells 3 x 10

⁹

bp - 10

⁶

fragments - plasmid, phage - 1-10 fragments

6. Isoschizomers - different MO and enzyme --> same cut site, but may generate different type of cut

For recombinant DNA work - develop plasmids that have a single RE site.

Restriction Mapping

Method to determine where a RE cuts DNA relative to other RE

Reasons for Isolating Random Genomic DNA fragment (blunt end fragments) - RE may cut inside the gene

- Methods - hydrodynamic shearing – vortexing, syringe needle

Joining of DNA Molecules/With Blunt Ends

A. Homopolymer tailing

-terminal nucleotidyl transferase, adds nucleotides to 3'-0H on SS-DNA, requires no template

-results in poly A and poly T tails

B. Blunt End ligation

T4 Ligase - will joint blunt ends, requires 3'-OH and 5'P DNA and ligase must be in high conc.

C. Linkers - short synthetic DNA fragments

contains a RE site --> attached with T4 ligase – often changes end to RE sticky end when digested with appropriate RE

Generate DNA Sequences (genes) that are hard to isolate:

1. Isolation of cDNA using Reverse Transcriptase (RT)

a. eukaryotic DNA cannot be put into prokaryotic cells --> mRNA will not be edited b. find eukaryotic cells that produce abundance of mRNA

c. isolate mRNA (already edited)

d. use RT isolated from cancer viruses (retroviruses - HIV) to make a DNA copy (termed cDNA)

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2. Amino acid sequencing of protein to be manufactured produce synthetic DNA (preferred codon program) clone/express sDNA

3. Formation of cDNA library mRNA with exons removed

library composed of coding regions search for genes (proteins) of interest

Detection of Recombinant Molecules

Possible products of ligation (cloning) reaction:

1. re-ligated vector

2. vector with one or more foreign DNA fragments 3. foreign DNA with no vector

Methods must address or differentiate between:

1. vector contains foreign DNA

2. foreign DNA contains gene of interest a. easy if gene is a selectable marker

b. difficult if eukaryotic gene does not change phenotype Common Methods used to Detect Clones/Transformed Cells 1. Insertional Inactivation

2 or more antibiotic resistant genes (pBR322)

Cycloserine enrichment technique - removes 1 step in the detection procedure 2. Cohesive ends, join 3'-OH and 5'P

-treat vector with alkaline phosphatase to remove 5'-P -prevent re-circularization of vector

-foreign DNA then supplies the 5'-P

3. Cosmids - plasmids that carry the COS site from λ phage

uses cos site-cutting system --> package DNA into phage head.

System only works on DNA if:

1. DNA must have 2 cos sites

2. cos sites separated by 38 kb to 54 kb packages DNA in vitro

-results in cloned DNA inserted between 2 cos sites -when cosmid is inserted into a cell - acts like a plasmid 4. α- complementation (lac Z method)

inactivation of lac Z gene codes for β-galactosidase

x-gal --> spread on plate surface, when metabolized results in a blue color Lac

⁺

--> blue colonies

Lac

^-

--> white colonies

system used in original automated DNA sequencing methods, cloning kits

Ways to get DNA into Target Cells

1. Transformation, Electroporation, Transduction

2. Liposomes

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-small spheres made from artificial membranes -fuse with plasma membranes

-release contents into the cell 3. Microinjection

-very fine needle to puncture cell wall/membrane -even directly into the nucleus (egg)

4. “Gene gun” – thick cell wall of plants

-particle of gold/nanosphere of silicon coated with DNA

Identifying Clones/Products after the DNA has Been Inserted into the Cell

1. Identify Recombinants by size -isolate plasmids and run a gel -select cultures with larger plasmids 2. Colony or In situ hybridization assay

need a probe - mRNA etc

3. Radioactive Antibody Test - test for protein production 4. Immunoprecipitation Test - test for protein production

temperature sensitive autolysis - cells lysed --> released protein protein reacts with antibody in agar plate

5. Epitope tags

-short DNA sequences fused in the ORF to be expressed -code for peptide recognized by commercial antibody -allow to identify/purify product

PCR - Polymerase Chain Reaction

Kary Mullis - history of discovery (Scientific American, April 1990) Requirements:

1. thermal cycler 2. primers

-complimentary to 3' end of DNA on either end of gene of interest -their 5' ends serve as primers for Tag polymerase

3. dNTPs

4. Taq Pol - not inactivated by denaturing temperature of cycler

Uses;

1. identify rare genes

2. recover genes in samples that are in low concentration

viral DNA sequences → detection of food poisoning MO (dead) 3. Site directed mutagenesis (change base in primer to induce mutation) 4. environmental surveys – non-culturable MO, metagenomics

5. used in DNA sequencing technology

6. make copies of specific genes for sequencing (16S rRNA genes)

Forensic Genetics using VNTR’S - Variable Number Tandem Repeats

cut w/RE

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gel w/fragments probe

autoradiography

“DNA” fingerprint STR’s - microsatellite repeats

microsatellite repeats - 30-35bp

Other Uses of Recombinant DNA Technology

exponential leap → prokaryotic system to eukaryotic system - cloning Euk genes in Prok cells