CHAPTER 9 DNA Technologies

Recombinant DNA

• Artificially created DNA that combines sequences that do not occur together in the nature

• Basis of much of the modern molecular biology

– Molecular cloning of genes

– Over-expression of proteins

– Transgenic food, animals …

DNA Cloning

• Organism cloning: – Creation of identical copies of an organism

• DNA cloning: – Creation of identical copies of a piece of DNA (gene) – Isolate a specific gene from the source organism and

amplify it in the target organism

• Basic steps: – Cut the source DNA at the boundaries of the gene – Select a suitable carrier DNA (vector) – Insert the gene into the vector – Insert the recombinant vector into host cell – Let the host produce multiple copies of recombinant DNA

DNA Cloning: General Scheme

• A cloning vector and eukaryotic chromosomes are separately cleaved with the same restriction endonuclease. (A single chromosome is shown here for simplicity.) The fragments to be cloned are then ligated to the cloning vector. The resulting recombinant DNA (only one recombinant vector is shown here) is introduced into a host cell, where it can be propagated (cloned). Note that this drawing is not to scale: the size of the E. coli chromosome relative to that of a typical cloning vector (such as a plasmid) is much greater than depicted here.

DNA Cloning: Generate Recombinant Vector

DNA Cloning: Introduce DNA into Organism

Cleavage of DNA molecules by restriction endonucleases. Restriction endonucleases recognize and cleave only specific sequences, leaving either (a) sticky ends (with protruding single strands) or (b) blunt ends. Fragments can be ligated to other DNAs, such as the cleaved cloning vector (a plasmid) shown here. This reaction is facilitated by the annealing of complementary sticky ends. Ligation is less efficient for DNA fragments with blunt ends than for those with complementary sticky ends, and DNA fragments with different (noncomplementary) sticky ends generally are not ligated. (c) A synthetic DNA fragment with recognition sequences for several restriction endonucleases can be inserted into a plasmid that has been cleaved by a restriction endonuclease. The insert is called a linker; an insert with multiple restriction sites is called a polylinker.

Restriction Endonucleases

• Cleave DNA phosphodiester bonds at specific sequences

• Common in bacteria

– Eliminates infectious viral DNA

• Some make staggered cuts

– Sticky ends

• Some make straight cuts

– Blunt ends

• Large number are known

– Commercially available

– Well-documented: REBASE

– http://rebase.neb.com/rebase/rebase.html

Restriction Endonucleases

Cloning Vectors

• Plasmids – Circular DNA molecules that are separate from the

bacterial genomic DNA – Can replicate autonomously

• Origins of replication for use in bacteria and/or yeast

– Carry antibiotic resistance genes – Allows cloning of DNA up to 15,000 bp

• To clone whole chromosomes (up to 300,000 bp)

– Bacterial Artificial Chromosome (BAC) • For use in bacteria

– Yeast Artificial Chromosome (YAC) • For use in yeast

Cloning Vectors: Plasmid

Typical Expression Vector

• DNA sequences in a typical E. coli expression vector. The gene to be expressed is inserted into one of the restriction sites in the polylinker, near the promoter (P), with the end of the gene encoding the amino terminus of the protein positioned closest to the promoter. The promoter allows efficient transcription of the inserted gene, and the transcription-termination sequence sometimes improves the amount and stability of the mRNA produced. The operator (O) permits regulation by a repressor that binds to it. The ribosome-binding site provides sequence signals for the efficient translation of the mRNA derived from the gene. The selectable marker allows the selection of cells containing the recombinant DNA.

Cloning Vectors: BAC Bacterial artificial chromosomes (BACs) as cloning

vectors. The vector is a relatively simple plasmid, with a replication origin (ori) that directs replication. The par genes, derived from a type of plasmid called an F plasmid, assist in the even distribution of plasmids to daughter cells at cell division. This increases the likelihood of each daughter cell carrying one copy of the plasmid, even when few copies are present. The low number of copies is useful in cloning large segments of DNA because it limits the opportunities for unwanted recombination reactions that can unpredictably alter large cloned DNAs over time. The BAC includes selectable markers. A lacZ gene (required for the production of the enzyme β-galactosidase) is situated in the cloning region such that it is inactivated by cloned DNA inserts. Introduction of recombinant BACs into cells by electroporation is promoted by the use of cells with an altered (more porous) cell wall. Recombinant DNAs are screened for resistance to the antibiotic chloramphenicol (CmR). Plates also contain X-gal, a substrate for β-galactosidase that yields a blue product. Colonies with active β-galactosidase and hence no DNA insert in the BAC vector turn blue; colonies without β-galactosidase activity—and thus with the desired DNA inserts—are white.

Cloning Vectors: YAC

Construction of a yeast artificial chromosome (YAC). A YAC vector includes an origin of replication (ori), a centromere (CEN), two telomeres (TEL), and selectable markers (X and Y). Digestion with BamHI and EcoRI generates two separate DNA arms, each with a telomeric end and one selectable marker. A large segment of DNA (e.g., up to 2 x 106 bp from the human genome) is ligated to the two arms to create a yeast artificial chromosome. The YAC transforms yeast cells (prepared by removal of the cell wall to form spheroplasts), and the cells are selected for X and Y; the surviving cells propagate the DNA insert.

DNA Ligase •Enzyme that covalently joins two DNA fragments

– Normally function in DNA repair

– Human DNA ligase uses ATP

– Bacterial DNA ligase uses NAD

DNA Ligase

Antibiotic Selection •Enzyme that covalently joins two DNA fragments

– Normally function

in DNA repair

– Human DNA

ligase uses ATP

– Bacterial DNA

ligase uses NAD

NH

S

N

N

O

O

H

OOH

HH

AMP

Identification of Empty Plasmids

Separation of DNA by Electrophoresis

• Negatively charged DNA migrates to the anode in the presence of an electric field

• Agarose gel hinders the mobility of DNA molecules

• Mobility depends on the size and the shape

– Small molecules faster

– Compact molecules faster

• Practical use

– DNA analysis

– DNA purification

– DNA-protein interaction studies

Expression of Cloned Genes

• We want to study the protein product of the gene

• Special plasmids, called expression vectors, contain

sequences that allow transcription of the inserted gene

• Expression vectors differ from cloning vectors by having:

– Promoter sequences

– Operator sequences

– Code for ribosome binding site

– Transcription termination sequences

Purification of Recombinant Genes

• Purification of natural proteins is difficult • Recombinant proteins can be tagged for purification • The tag binds to the affinity resin, binding the

protein of interest to a purification column

Purification of Recombinant Genes The GST tag is fused to the

carboxyl terminus of the protein by genetic engineering. The tagged protein is expressed in the cell and is present in the crude extract when the cells are lysed. The extract is subjected to affinity chromatography through a matrix with immobilized glutathione. The GST-tagged protein binds to the glutathione, retarding its migration through the column, while the other proteins are washed through rapidly. The tagged protein is subsequently eluted with a solution containing elevated salt concentration or free glutathione.

Polymerase Chain Reaction (PCR)

• Used to amplify DNA in the test tube – Can amplify regions of interest (genes) within linear DNA – Can amplify complete circular plasmids

• Mix together – Target DNA – Primers (oligonucleotides complementary to target) – Nucleotides: dATP, dCTP, dGTP, dTTP – Thermostable DNA polymerase

• Place the mixture into thermocycler – Melt DNA at about 95°C – Cool separated strands to about 50–60°C – Primers anneal to the target – Polymerase extends primers in 5’3’ direction – After a round of elongation is done, repeat steps

General Steps of PCR

• Amplification of a DNA segment by the polymerase chain reaction (PCR). (a) The PCR procedure has three steps. DNA strands are 1 separated by heating, then 2 annealed to an excess of short synthetic DNA primers (orange) that flank the region to be amplified (dark blue); 3 new DNA is synthesized by polymerization catalyzed by DNA polymerase. The three steps are repeated for 25 or 30 cycles. The thermostable Taq DNA polymerase (from Thermus aquaticus, a bacterial species that grows in hot springs) is not denatured by the heating steps.

Repeat steps 1–3 many times: After 25 cycles DNA has been amplified about 106 fold

Adaptations to PCR

• qReverse Transcriptase PCR (RT-PCR)

• Real-Time Quantitative Reverse Transcription PCR

• www.ncbi.nlm.nih.gov › ProbeDB › Technologies

– Used to amplify RNA sequences

– First step uses reverse transcriptase to convert RNA to DNA

Cells in all organisms regulate gene expression by turnover of gene transcripts (messenger RNA, abbreviated to mRNA): The amount of an expressed gene in a cell can be measured by the number of copies of an mRNA transcript of that gene present in a sample. In order to robustly detect and quantify gene expression from small amounts of RNA, amplification of the gene transcript is necessary. The PCR is a common method for amplifying DNA; for mRNA-based PCR the RNA sample is first reverse-transcribed to cDNA with RT.

• Quantitative PCR (Q-PCR)

– Used to show quantitative differences in gene levels

– Quantitative PCR and DNA microarray are modern methodologies for studying gene expression.

Quantitative PCR. PCR can be used quantitatively, by

carefully monitoring the progress of a PCR amplification and determining when a DNA segment has been amplified to a specific threshold level. (a) The amount of PCR product present is determined by measuring the level of a fluorescent probe attached to a reporter oligonucleotide complementary to the DNA segment that is being amplified. Probe fluorescence is initially not detectable due to a fluorescence quencher attached to the same oligonucleotide. When the reporter oligonucleotide pairs with its complement in a copy of the amplified DNA segment, the fluorophore is separated from the quenching molecule and fluorescence results. (b) As the PCR reaction proceeds, the amount of the targeted DNA segment increases exponentially, and the fluorescent signal also increases exponentially as the oligonucleotide probes anneal to the amplified segments. After many PCR cycles, the signal reaches a plateau as one or more reaction components become exhausted. When a segment is present in greater amounts in one sample than another, its amplification reaches a defined threshold level earlier. The “No template” line follows the slow increase in background signal observed in a control that does not include added sample DNA. CT is the cycle number at which the threshold is first surpassed.

Eukaryotic Gene Expression in Bacteria

• An eukaryotic gene from the eukaryotic genome will

not express correctly in the bacterium

• Eukaryotic genes have:

– Exons: coding regions

– Introns: noncoding regions

• Introns in eukaryotic gene pose problems

• Bacteria cannot splice introns out

• mRNA is intron-free genetic material

Construction of cDNA

• mRNA can be extracted from eukaryotic cells

• All mRNA molecules have

poly-A tail

– Helps in purification of mRNA

– Serves as a universal template

• DNA strand can be synthesized using mRNA as a template

• This is catalyzed by the reverse transcriptase

• The end result is a hybrid where the DNA strand is complementary to the mRNA

• The hybrid can be converted to duplex DNA, known as cDNA

Construction of cDNA

• Building a cDNA library from mRNA. A cell’s total mRNA content includes transcripts from thousands of genes, and the cDNAs generated from this mRNA are correspondingly heterogeneous. Reverse transcriptase can synthesize DNA on an RNA or a DNA template. To prime the synthesis of a second DNA strand, oligonucleotides of known sequence are ligated to the 3’ end of the first strand, and the double-stranded cDNA so produced is cloned into a plasmid.

Construction of cDNA

Construction of cDNA

Site-Directed Mutagenesis

Site-Directed Mutagenesis

• Understanding the function of proteins often requires that a specific amino acid residue be mutated

• To mutate an amino acid, change the nucleotide(s) in the coding DNA and express the mutated gene

• Site-directed mutagenesis usually relies on chemically synthesized mutated primers that are incorporated into newly synthesized DNA

• Mutated plasmids are always sequenced to confirm the desired (and only the desired) mutation is present

Fluorescence can be used to determine protein location in vivo

• Green fluorescent protein (GFP)

– Use recombinant DNA technologies to attach GFP to protein of interest

– Visualize with a fluorescent microscope

• Immunofluorescence

– Tag protein with primary antibody and detect with secondary antibody containing fluorescent tag

– Protein can also be fused to a short epitope and the primary antibody detecting the epitope can be fluorescently labeled

GFP–Tagged Protein Localization

Immunofluorscence

Visualization of protein location from a GFP–Tagged cDNA Library

Identifying Protein–Protein Interactions

• Protein complex isolation

– Epitope tag one protein in the complex

– Gentle isolation of epitope-tagged protein will also isolate stably interacting proteins

– All proteins isolated can be separated and identified

– Use of Tandem Affinity Purification (TAP) tags has enhanced the procedure

• Allows two purification steps eliminating loosely associated proteins, and minimizing non-specific binding

Identifying Protein-Protein Interactions

• The use of epitope tags to study protein-protein interactions. The gene of interest is cloned next to a gene for an epitope tag, and the resulting fusion protein is precipitated by antibodies to the epitope. Any other proteins that interact with the tagged protein also precipitate, thereby helping to elucidate protein-protein interactions.

Procedure for TAP–Tagged Proteins