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A little history

•  Up until the 1970’s DNA was actually one of the hardest things to analyze and characterize.

•  Currently it is probably one of the easiest things to work with and characterize, can sequence a whole genome, millions of bp per day

•  Currently we can sequence DNA, engineer DNA and manipulate DNA in a variety of ways. Can know mutations for specific conditions and target treatments

1

Polymerase Chain Reaction

•  Method to amplify a specific fragment of DNA

•  Also based on in vitro DNA synthesis

•  Has many different uses - revolutionized molecular biology

Cloning a it of DNA to make millions of copies

2

How did PCR get invented?

•  Kary Mullis thought about it on a car ride up the coast

•  Earned Nobel Prize in Chemistry in 1993

3

PCR primers

Oligonucleotide primers which bound theregion to be amplified are made

20-25 bp, finding exact same match in other places in the DNA is impossible

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PCR Polymerase

Taq polymerase, isolated form

Thermus aquaticus, is resistant to heat denaturation

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PCR: The Reaction

Repeated many times to amplify a specific region from as little as one copy of DNA

•  Three basic steps:
–  Denaturation (92 degrees)
–  Hybridization (annealing temp 50-58 degrees)
–  Elongation (72 degrees)

•  This is repeated over and over for exponential “growth” ofthe amplified fragment

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What makes a good primer?

20-25 bp

1/2 GC content

Tm = 2(A+T) + 4(G+C), should be about 50-58 degrees (below 72 degrees or primer will come off)

G/C at 3' end for elongation

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How many cycles are in a PCR reaction?

The process is repeated 25-35 times, to yield over 1 billion copies of the amplified region from a single copy of DNA

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What else is in a PCR reaction?

Buffer- if higher salt content, easier primer match

Magnesium chloride- also controls permissiveness

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Application of PCR

•  Cloning genes
•  Diagnosis of inherited diseases
•  Detection of viruses (HIV)
•  Studies of gene expression during development
•  Forensics (DNA fingerprinting)
•  Evolution (amplification of DNA of extinct species)

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Recombinant DNA technology

Utilizes a mixture of new techniques and some borrowed from other fields, Some of the techniques are utilized in a variety of different ways

–  Restriction nucleases
–  DNA cloning
–  Nucleic acid hybridization
–  Sequencing of nucleotides in a purified DNA fragment
–  Monitoring gene expression

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Why isolate or clone a gene?

•  To study how genes and proteins function in the cell and how cells function in an organism
–  When and where is a gene expressed?
–  How is gene expression regulated?
–  How does a gene act to make a cell function?
–  How are individual genes involved in a particular disease?

•  To make therapeutical proteins (recombinant protein drugs)

•  To replace the “bad” gene in a patient (gene therapy)

•  To improve agricultural live stock and crops (GM foods)

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We have the DNA but want to make more copies

•  The best way to clone a DNA is to have the cell replicate it

•  Cells won’t replicate just a fragment of DNA

•  The fragment of DNA needs to be placed (ligated or inserted) into a vector that can undergo replication…fragment of DNA + vector = recombinant DNA

•  A vector is a self-replicating DNA (i.e. plasmid or virus)

•  Need a host that can uptake and replicate the recombinant DNA (i.e. E. coli and transformation)

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Features of a Vector

(1)  a replicon: origin of replication

(2) a selectable marker: allows for selection;
- is often a bacterial gene that confers antibiotic resistance
- some of the often used antibiotic resistance are ampr, kanr, tetr, etc.

(3) a polylinker: is a small region of DNA that contains multiple unique restriction sites that are not found elsewhere , where restriction enzyme will cut
- Known site where foreign DNA are inserted (ligated)

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Forming Recombinant DNAs

When restriction fragments are combined with a vector we have engineered a recombinant DNA. Cut with same restriction enzyme to have same sticky ends

Vector - a DNA molecule that replicates independent of the genome

Insert - DNA that is introduced into the vector

DNA Ligase

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Plasmids

•  Circular double stranded DNA

•  These occur naturally in bacteria yeast, and higher eukaryotes

•  Can be parasitic or symbiotic with their hosts

•  Replicate separately from the chromosomal DNA of the cell

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What are the requirements to use plasmids as vectors?

–  Selectable marker

–  Must be able to introduce the recombinant DNA into the cell (process called transformation)

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Restriction enzymes

Homodimer, recognizes palindromic sequences

Some create blunt ends, some create sticky ends

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How do we find just the gene we are looking for?

•  You need to “fish” the right gene out of all of your recombinant fragments

•  The fragment or gene of interest is also needed in large quantities.

•  DNA cloning is a technique to produce these large quantities (it is also used for other purposes)

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How do the plasmids/vectors enter the bacterial cells?

Recall that Avery, Macleod and McCarty showed bacterial cellstake up DNA from their surroundings (transformation)

Two common methods:
Salt (CaCl2)
Electroporation- metal plate with current, makes hole in cell wall so plasmid can enter

Sometimes EColi have plasmid without DNA of interest

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Process of inserting a gene

Purify human and plasmid DNA

Cut with same enzyme and join fragments into recombinant DNA with ligase (don't have to use the same enzyme, just ahve to have same sticky ends)

Incubate E.Coli so that they take up the plasmids, and grow them in a medium that selects for those containing a recombinant plasmid

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Selectable marker

•  Any gene whose product confers unique properties tothe host cell
–  These can be products so that cells only grow under certain conditions
–  These can be genes whose products allow cell growth in the presence of inhibitors

•  Ampicillin - inhibits cell wall synthsesis

•  Tetracycline - Inhibits protein synthesis by binding to 30S ribosomal subunit and interferes with the binding of tRNA to mRNA

Only cells with plasmid grow

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If the cell is growing, does that mean it has the plasmid of interest?

No, just means it has plasmid with selectable marker (usually resistance to something)

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How do you find plasmid with DNA of interest?

Replica plating

Screening colonies

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Replica plating

Stamp the colonies on the plate repeatedly with sterile paper ("velvet") which pulls up a couple cells from each colony, doesn't destroy colony

Make sure you know orientation of colonies on new plate

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Screening colonies

Culture dish with bacterial colonies (or phage plaques) containing recombinant DNA

Transfer representative cells to nitrocellulose membrane by replica plating technique

Lyse cells and treat to denature DNA and cause single stranded DNA to adhere to the filter in place

Denatured DNA adhering to the membrane

Incubate with radioactive probe using what you know about the gene (promoter sequence etc) and make autoradiograph

Silver grains on x-ray film show position of labeled hybrid (or can use immunoflouresence)

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Phage and viruses as vectors

Commonly used, can take up larger DNA inserts than plasmids

Bacteriophage λ is one of the most commonly used vectors. A non-essential region in the middle is deleted and replaced by insert DNA.

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How is phage lamda modified for use as a cloning vector?

Genes for the lysogenic state are not essential for lytic growth, replace them with DNA of interest

•  Can replace with up to ~25kb of exogenous DNA (may or may not get enhancer sequences)

•  Need to repackage into virions

•  Phage lambda is very efficient (infects and replicates well)

28

Lytic vs Lysogenic

Viruses very good at replicating dna two states.

lytic: Takes hold of replication machinery of host cell and creates thousands of viral genomes, proteins, everything virus needs is generated. Bursts the cell and viruses released

Lysogenic: Incorporate their genome into the host genome and will only replicate when cell DNA replicates, became part of DNA chromosome

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Assembling a genomic library in Phage lambda

•  Cleave genomic DNA into 20kb fragments with Sau3A

•  Remove replaceable region of phage genome with BamHI (not same enzyme but same sticky ends)

•  Ligate lambda arms to genomic DNA

•  Package in vitro

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Max size of DNA that can be cloned on vectors

Plasmid- 20kb
Bacteriophage lambda- 25kb
Cosmid- 45kb
P1 vector- 100kb
Yeas artificial chromosome- 1000kb

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How do you find the DNA of interest in the phages?

There is a bacterial lawn on a culture dish

Layer of bacterial cells covering the plate, small dilution of phage put on plate. Few phage but wherever infects the first host cell, its gonna burst out and infect cells around original cell. Because in lytic cycle, bursts those. Not opaque bacterial lawn anymore. Clear circle called plaques that contain phage particles. Can investigate each plaque to find DNA of interest

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Vectors used in making DNA library

Phage, cosmid, BAC, YAC

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Phage (lambda)

phage genomes are usually linear DNA, and thoseused for vector are often extensively modified. Can hold larger DNA
inserts than plasmids.

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Cosmid

hybrid of plasmid and phage. It is circular vector and replicates as a plasmid, and can also be packaged as phage particles in vitro.

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BAC(bacterial artificial chromosome)

specialized bacterial plasmids (F factors), Ori, and gene encoding Ori-binding protein, so it replicatelike a plasmid but can hold much more DNA.

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YAC (yeast artificial chromosome)

linear DNA vector containing theyeast origin of replication, centromere sequence for chromosome separation, and telomere sequence (at both ends). YAC are eukaryote plasmid.

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Cloning specific fragments

•  Less than 5% of the genome is expressed- can see what is expressed without the introns

•  Tissue specificity

•  cDNA = copy or complementary DNA derived from mRNA

•  Only exons are represented - can determine complete openreading frame of a gene

•  Only expressed mRNAs will be cloned - choice of cell type is important

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How do we isolate the mRNAs?

•  Only ~3% of cellular RNA is mRNA

•  Need to isolate from rRNA and tRNA etc.

•  Only mRNAs have poly-A tails.

•  Poly-A anneals to oligo dT.

•  mRNAs selectively bind to oligo dT resin.

•  After binding RNA to the resin, tRNA and rRNA are washed out.

•  mRNA is then elutedand used for cloning or other analysis

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Reverse transcriptase

allows the conversion of RNA sequence into DNA

•  RNA dependent DNA polymerase

•  Enzyme isolated from retrovirus virions

•  Needs a primer and an RNA or DNA template

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How do you get the cDNA?

Anneal poly(dT) primer to poly(A) tail of mRNA

Make DNA copy with reverse transcriptase

Treat hybrid with RNase H,which nicks the RNA, leaving free 3'-OH groups

Add DNA polymerase I which uses the RNA fragments as primers and replaces the RNA with DNA

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What are the advantages of having a cDNA library?

You see directly the genes/sequences that code for proteins

However, since its the exon sequences only, you may not have everything that expresses the functional protein (promoters, translational gene expression, etc)

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methods commonly used to identify a clone of interest from a library

1  Select for expression of cloned gene in a mutant cell background (common techniqueused in bacteria or yeast)

2  Purify the protein and use it to identify the gene (either through an antibody probe or an oligonucleotide probe).

3  Hybridization with a probe (DNA or RNA fragment as a probe because they can form double stranded hybrids)

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expression of cloned gene in a mutant cell background

Make plasmid (ampr) library using E. coli genomic DNA, inserting the DNA of interest into the lac gene

Plate on minimal lactose media + ampicillin (selecting for Lac+/ampr)

Only bacteria that took up a plasmid will grow but we don't know whether or not an insert is in a plasmid, so we see which colonies cleave when exposed to xgal and iptg

Colonies that you screen for insert of interest is white colonies because insertion site for library fragments is in the middle of the lac operon. If plasmid took in an insert it made the operon non functional so xgal cannot be cleaved

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isolate the protein using standard biochemical techniques

1  Identification of the gene using an antibody against the purified protein (expression cloning)

2  Identification of the gene using the sequence ofthe protein to synthesize an oligonucleotide probe (hybridization)

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Expression Cloning

Requires special vectors, (also needs selectable marker and origin)

Cloned DNA is transcribed and translated in cell

Coding sequence (usually cDNA) is inserted in frame into a gene of the vector (Plasmids inserted right next to a promoter. Cut site at 3' end of the promoter

Creates a fusion protein present incolony or plaque (Gene inserted in reading frame with known part of protein. For that known part, we can isolate it using an antibody that binds to the known part of the protein and isolate it)

Screen with an antibody or other molecule known to bind to the protein.

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Antibody screen

•  Vector: bacteriophage expression vector
–  When the phage lyse the cells, they release theexpressed protein in the plaque, which can be detected using antibodies.

•  Inserts:
–  cDNA-eukaryotes,
–  genomic-prokaryotes

•  Probe: antibody raised against protein of interest

•  Screen: plaque hybridization

•  Detection: labeled secondary antibody

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Plaque hybridization

Proteins are transferred to a nitrocellulose filter and probed with antibody specific for the protein of interest. The antigen antibody complexes are then detected with a secondary labeled antibody.

Only plaques with protein of interest are sticking to antibodies. Link to second antibody with alkaline phosphatase for color change. Know where DNA of interest is because it made protein of interest

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Hybridization with a probe

Purified protein (don't know where sequences are in the chromosome), do Edman degradation and find amino acid sequence from N to C

Sequence a protein fragment

Use genetic code to determine all possible coding sequences

Synthesize oligonucleotides to be used as a probe

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How do you make the probe?

Many possible probes to make because many codons in codon table

Don't wanna use a region of the protein that gives you many possible probes. Search for region of protein that is less degenerate. When doing Edman degradation, want 6/7 amino acids, gives you probe that is 18-21 bases long

Flourescently labeled or radioactivity, put them in a tube and then hybridize. Radioactivity- use kinase, add radiactive phosphate group

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Preparation of radioactive probes

1 - Phosphatase removes 5’ phosphate from DNA restriction fragment (not necessary for oligonucleotide)

2 - Polynucleotide kinase adds 32P-phosphate to the 5’ end of a DNA chain

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Hybridization process

Take dna containing gene of interest, heat it up. Then denature it , then mix it with oligonucleotide probes

All could be compimentary to proteins, but only one will be the exact sequence for the protein. After denaturing you will reanneal and hybridize the probe. Now you can see where the sequence is on a chromosome. Can also go back to nitrocellulose filter and affix the plaques to the nitrocelluluse and use probe to identify which has insert of interest.

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Screening libraries using other DNA orRNA probes

Don't have to just use edmund degradation to get the probes.

•  Possible probes:
– cloned gene from another species
– cDNA can be used as probe to isolate the genomic clone (Use known dna to probe a library to check for same sequencies between species, would want less stringent conditions, more permissive. (not too low of salt, not too high of temperature))
– RNA

•  Plaque or colony hybridization