recombinant dna and biotechnology · 2020. 11. 9. · recombinant dna and biotechnology...
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Lecture 23 (11/9/20) Nucleic AcidsA. The 4 S’s
1. Size2. Solubility3. Shape
a. A-DNAb. Z-DNAc. Topology
i. Packagingii. Supercoilingiii. Topoisomerases
4. Stabilitya. Nucleotides
i. Tautomersii. Acid/base
b. Nucleic Acidsi. Chemistryii. Denaturationiii. Stabilityiv. Nucleases
B. Structure of the Information1. Exceptions to flow2. Structure3. Levels of Control
C. Recombinant DNA: Biochemical Basis of Biotechnology
1. Restriction enzymes, DNA ligase2. Vectors and Inserts to make
recombinant DNA (rDNA)3. Transformation of hosts4. Selection of transformants5. Expression6. Site-directed mutagenesis
• Reading: Ch1; 29-34Ch8; 295-299Ch9; 319-325, 346
• Problems: Ch8 (text); 6,7,8,10Ch8 (study-guide: applying); 1,3Ch8 (study-guide: facts); 10,11Ch9 (text); 1,2,3,4Ch9 (study-guide: facts); 1,2,3,4,5Ch24 (study-guide: facts); 3,5,6Ch26 (text); 3Ch26 (study-guide: applying); 2,3Ch26 (study-guide: facts); 7Ch27 (text); 1,2,3,4
NEXT•Reading: Ch9; 328-332
Ch25; 990-995, 1005-1012
•Problems: Ch9 (study-guide: applying); 1,2Ch9 (study-guide: facts); 7,8Ch25 (text); 1-3,5-7,9,10,13-15Ch25 (study-guide: applying); 1,4Ch25 (study-guide: facts); 3,4,6
Recombinant DNA and Biotechnology
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Nucleicacidfunction:CentralDogma
prokaryotes eukaryotes
Coding Region5' Flanking 3'-flanking
The structure of the information:
Promoter
prokaryotes
GENE:
mRNA:
PROTEIN:
+1
eukaryotes–10–35
TATA boxOther elements
Teminator
Exon 1 Exon 2Intron 1
Start of Transcription
End of Transcription
prokaryoteseukaryotes
Start of Translation
End of Translation
AUGStopCodon
OPEN READING FRAME(ORF)
5’-UTR 3’-UTRShine-Delgarno
5’-cap 3’-Poly(A)
Spliced
C-TermN-Term
Example:
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Nucleicacidfunction:CentralDogma
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)
• RNA editing –change in ORF not from DNA
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• Reverse transcription –making DNA from RNA (retroviruses)
Used to make copy DNA (cDNA), which is a copy of the mRNA
RTCase
Control of Gene Expression: EukaryotesEukaryotic gene regulation can occur at multiple points in transcription and translation:•Initiation of transcription
wActivation of transcriptional initiationwChromatin remodeling
•mRNA processing•mRNA transport•mRNA stability•Initiation of translation•Post-translational controls•Protein stability
prokaryotes eukaryotes
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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
GENEPROTEIN
Biochemistry Genetics
recDNA
Recombinant DNA has one simple goal: MAKE MORE
Recombinant ProteinsRecombinant DNA and Biotechnology
………coronavirus
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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
Recombinant DNA is DNA made in the laboratory that is derived from at least two genetic sources.
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.
Others you will see how they are used when we go through the processes.
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Restriction SitesRecombinant DNA and Biotechnology
Restriction enzymes recognize palindromic DNA sequences:
5ʼ…….GAATTC……3ʼ3ʼ…….CTTAAG……5ʼ
Some make straight cuts, others make staggered cuts, resulting in overhangs or sticky ends.
EXAMPLES:
5ʼ…….G-3ʼ 5ʼ-AATTC……3ʼ3ʼ…….CTTAA-5ʼ 3ʼ-G……5ʼ
Restriction SitesRecombinant DNA and Biotechnology
Restriction enzymes recognize palindromic DNA sequences:
5ʼ…….GAATTC……3ʼ3ʼ…….CTTAAG……5ʼ
Some make straight cuts, others make staggered cuts, resulting in overhangs or sticky ends.
EXAMPLES:
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EcoRVEndonuclease
EcoRV endonucleasePDBid 4RVE
EcoRIEndonuclease
EcoRI endonucleasePDBid 1ERI
Restriction EndonucleasesRecombinant DNA and Biotechnology
Restriction Endonucleases Cleave at Specific Recognition Sites
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DNALigasewill“seal”the“nick”bymakingthecovalentphosphodiesterbond
Restriction Endonucleases Key to Making rDNA Molecules
DNA Ligase: Also Key to Making rDNA Molecules
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DNA Ligase Reaction
Process Diagram:Recombinant DNA Construction
Recombinant DNA molecule
Restriction Endonucleases & DNA Ligase: Key to Making rDNA Molecules
Where does this Foreign DNA come from and how is it purified?
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A genomic clone A cDNA clone
Vectors and InsertsRecombinant DNA and Biotechnology
A genomic clonecontains 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.
Represents an mRNA from a given cell/tissue. cDNA is produced by making a DNA copy of the mRNA population using the RNA-directed DNA polymerase called, reverse transcriptase.
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
DNA fragments used for molecular cloning come from two sources:• Genomic DNA• cDNA (Copy DNA or complementary DNA)From reverse transcription of mRNA
A genomic clone A cDNA clone
Vectors and InsertsRecombinant DNA and Biotechnology
A genomic clonecontains 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.
Represents an mRNA from a given cell/tissue. cDNA is produced by making a DNA copy of the mRNA population using the RNA-directed DNA polymerase called, reverse transcriptase.
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
DNA fragments used for molecular cloning come from two sources:• Genomic DNA• cDNA (Copy DNA or complementary DNA)From reverse transcription of mRNA
Where does this Foreign DNA come from and how is it purified?
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11-11-2020
NO CLASS! Enjoy the day off.