Recombinant DNA and Biotechnology. Nucleic acid function: Central Dogma

Recombinant DNA and Biotechnology Nucleic acid function: Central Dogma 1 The structure of the information: 5' Flanking GENE: prokaryotes eukaryot...
Author: Derek Matthews
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Recombinant DNA and Biotechnology

Nucleic acid function: Central Dogma

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The structure of the information: 5' Flanking

GENE: prokaryotes eukaryotes

+1

–35

Teminator

Coding Region

Promoter

Exon 1

–10

Intron 1

3'-flanking

Exon 2

Other elements TATA box

Start of Transcription

mRNA: prokaryotes eukaryotes

AUG 5’-UTR Shine-Delgarno

End of Transcription

Example:

Stop Codon

OPEN READING FRAME (ORF)

3’-UTR 3’-Poly(A)

5’-cap

PROTEIN:

prokaryotes

Spliced

Start of Translation

N-Term

End of Translation

C-Term

eukaryotes

Nucleic acid function: Central Dogma

Exceptions to the direction of the information flow: • RNA editing – change in ORF sequence, but not from DNA • Reverse transcription – making DNA from RNA (retroviruses)

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• RNA editing – change in ORF not from DNA

• Reverse transcription – making DNA from RNA (retroviruses)

Used to make copy DNA (cDNA), which is a copy of the mRNA

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Control of Gene Expression: Eukaryotes

Eukaryotic gene regulation can occur at multiple points in transcription and translation: •Initiation of transcription wActivation of transcriptional initiation wChromatin remodeling

•mRNA processing •mRNA transport •mRNA stability •Initiation of translation •Post-translational controls •Protein stability

prokaryotes

eukaryotes

Recombinant DNA and Biotechnology Recombinant DNA is DNA made in the laboratory that is derived from at least two genetic sources. Recombinant DNA has allowed molecular biology to come full circle. FUNCTION Biochemistry

Genetics

PROTEIN

GENE rDNA

Recombinant DNA has one simple goal: MAKE MORE

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Recombinant DNA and Biotechnology Recombinant Proteins

Recombinant DNA and Biotechnology Recombinant DNA is DNA made in the laboratory that is derived from at least two genetic sources.

• Biochemical Basis of Biotechnology - Restriction enzymes, DNA ligase - Vectors and Inserts to make recombinant DNA (rDNA) - Transformation of hosts - Selection of transformants - Expression - Site-directed mutagenesis

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Recombinant DNA and Biotechnology

Restriction enzymes are used to cut DNA into fragments, which then are spliced together in new combinations. DNA ligase catalyzes the joining of DNA fragments.

Recombinant DNA and Biotechnology Restriction Sites Restriction enzymes recognize palindromic DNA sequences: 5ʼ…….GAATTC……3ʼ 3ʼ…….CTTAAG……5ʼ Some make straight cuts, others make staggered cuts, resulting in overhangs or sticky ends.

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Recombinant DNA and Biotechnology Restriction Endonucleases EcoRI Endonuclease

EcoRI endonuclease PDBid 1ERI

EcoRV Endonuclease

EcoRV endonuclease PDBid 4RVE

Restriction Endonucleases Cleave at Specific Recognition Sites

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Restriction Endonucleases Key to Making rDNA Molecules

DNA Ligase will “seal” the “nick” by making the covalent phosphodiester bond

DNA Ligase: Also Key to Making rDNA Molecules

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DNA Ligase Reaction

Restriction Endonucleases & DNA Ligase: Key to Making rDNA Molecules Process Diagram: Recombinant DNA Construction

Recombinant DNA molecule

Where does this Foreign DNA come from and how is it purified?

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Recombinant DNA and Biotechnology fragments used for molecular cloning come from two sources: Vectors and Inserts DNA • Genomic DNA • cDNA (Copy DNA or complementary DNA)From reverse transcription of mRNA

Represents an mRNA from a given cell/tissue. cDNA is produced by making a DNA copy of the mRNA population using the RNAdirected DNA polymerase called, reverse transcriptase.

A genomic clone contains the gene(s) as a fragment of the genome of an organism. The DNA is cut into fragments by restriction enzymes, and each fragment is inserted into a vector.

A genomic clone

A cDNA library is a “snapshot” of the transcription pattern of the cell. cDNA clones are used to provide the ORF for expressing the protein

A cDNA clone

Recombinant DNA and Biotechnology Vectors and Inserts: cDNA Cloning

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Recombinant DNA and Biotechnology Vectors and Inserts: cDNA Cloning

How do you find your DNA of interest? Restriction Digest Electrophoretogram

Process Diagram: Southern Blotting

Using polynucleotide kinase and g32P-ATP

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Recombinant DNA and Biotechnology • Biochemical Basis of Biotechnology - Restriction enzymes, DNA ligase - Vectors and Inserts to make recombinant DNA (rDNA) - Transformation of hosts - Selection of transformants - Expression - Site-directed mutagenesis All vectors have 3 things: 1. Autonomous replication ability 2. Selection for hosts that contain vector 3. Site for insertion of rDNA Viruses as vectors (bacteriophage or retroviruses). • Viruses can be altered to attenuate some detrimental genes in the virus (e.g., those that kill the host). • Viruses can be altered to carry recombinant DNA into cells

Recombinant DNA and Biotechnology • Biochemical Basis of Biotechnology - Restriction enzymes, DNA ligase - Vectors and Inserts to make recombinant DNA Inserting the recombinant DNA into a cell: (rDNA) • Cells may be treated with chemicals to make plasma membranes more - Transformation of hosts permeable—DNA diffuses into cells. - Selection of transformants • Electroporation—a short electric shock Transformation: Recombinant DNA is cloned creates temporary pores in membranes, - Expression by inserting it into host cells (transfection if and DNA shoots to the + end and can enter cells. Site-directed mutagenesis host-cells are from an animal). Second key discovery in biotechnology. Usually only a few cells are transformed (1 cell in 10,000). Reason for the need for a selectable marker. The first host cells used were bacteria, especially E. coli. Yeasts (Saccharomyces) are commonly used as eukaryotic hosts.

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Recombinant DNA and Biotechnology • Biochemical Basis of Biotechnology - Restriction enzymes, DNA ligase - Vectors and Inserts to make recombinant DNA (rDNA) - Transformation of hosts - Selection of transformants • Use ofExpression antibiotic resistance gene (e.g., ampicilin resistance) on a plasmid mutagenesis - Site-directed • For viral vectors, use of “infected” phenotype. • Use of “selectable markers” to detect either insertion into the vector or incorporation into the host. Some of these are a type of reporter gene—a gene whose expression is easily observed. • Many plasmids contain the lacZ gene with a multiple cloning site within its sequence. lacZ codes for an enzyme that can convert the substrate X-Gal into a bright blue product

Plac

Insert ORF of interest

• Origin of replication • Antibiotic resistance gene • Multiple cloning site • Promoter for transcription, translation (host specific)

Recombinant DNA Clone Ori

–Insert – Makes b-Galactosidase +Insert – Doesn’t make b-Galactosidase

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