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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.