Recombinant DNA Technology: PCR, Southern Blot, and Microarrays

Recombinant DNA Technology

Needles in Haystacks

· How to find one gene in large genome? A gene might be 1/1,000,000 of the genome. Three basic approaches:

• 1. Polymerase chain reaction (PCR). Make many copies of a specific region of the DNA.
• 2. cell-based molecular cloning: create and isolate a bacterial strain that replicates a copy of your gene.
• 3. hybridization: make DNA single stranded, allow double strands to re-form using a labeled (e.g. radioactive) version of your gene to make it easy to detect.

Polymerase Chain Reaction (PCR)

· Based on DNA polymerase creating a second strand of DNA. - Needs template DNA and two primers that flank the region to be amplified. Primers are short (generally 18-30 bases) DNA oligonucleotides complementary to the ends of the region being amplified. - DNA polymerase adds new bases to the 3' ends of the primers to create the new second strand. - go from 1 DNA to 2, then 4, 8, etc: exponential growth of DNA from this region - A key element in PCR is a special form of DNA polymerase from Thermus aquaticus, a bacterium that lives in nearly boiling water in the Yellowstone National Park hot springs. This enzyme, Tag polymerase, can withstand the temperature cycle of PCR, which would kill DNA polymerase from E. coli.

PCR Advantages and Disadvantages

· advantages: - rapid, sensitive, lots of useful variations, robust (works even with partly degraded DNA) · disadvantages: - Only short regions (up to 2 kbp) can be amplified. - limited amount of product made

PCR Cycle

· PCR is based on a cycle of 3 steps that occur at different temperatures. Each cycle doubles the number of DNA molecules: 25-35 cycles produces enough DNA to see on an electrophoresis gel. Each step takes about 1 minute to complete.

• 1. Denaturation: make the DNA single stranded by heating to 94℃
• 2. Annealing: hybridize the primers to the single strands. Temperature varies with primer, around 50℃
• 3. Extension: build the second strands with DNA polymerase and dNTPs: 72ºC.

Other PCR Images

DNA Amplification in PCR

· original DNA: very long molecules with neither end well defined. Number stays constant in the PCR reaction: no new ones are made.

• initial PCR product made from original DNA: has one end defined by the primer, but the other end is not well defined. Copy number grows linearly.

. all other PCR products have 2 ends defined by the primers, so they have a constant length and can be easily detected by electrophoresis. Copy number grows exponentially.

1. DNA is denatured. Primers attach to each strand. A new DNA strand is synthesized behind primers on each template strand. primer 2. Another round: DNA is denatured, primers are attached, and the number of DNA strands are doubled. Another round: DNA is denatured, primers are attached, and the number of DNA strands are doubled. Another round: DNA is denatured, primers are attached, and the number of DNA strands are doubled,

Continued rounds of amplification swiftly produce large numbers of identical fragments. Each fragment contains the DNA region of interest.

Electrophoresis

· Separation of charged molecules in an electric field. · Nucleic acids have 1 charged phosphate (- charge) per nucleotide. means constant chare to mass ratio. Separation based (mostly) on length: longer molecules move slower. · Done in a gel matrix to stabilize: agarose or acrylamide. · average run: 100 Volts across a 10 cm gel, run for 2 hours. · Stain with ethidium bromide: intercalates between DNA bases and fluoresces orange. · Run alongside standards of known sizes to get lengths

PCR Applications

· RT-PCR: use reverse transcriptase to convert messenger RNA into DNA, then amplify it with PCR.

• Anchor-primed PCR: use one sequence-specific primer and use a set of random primers for the other end. For example: 3' RACE-PCR (Rapid Amplification of cDNA Ends) uses an oligo-dT primer to bind to the poly A tail of mRNA and a universal primer for the internal region.

. Adding linkers to the primers puts them into the amplified DNA. Useful for cloning or further PCR.

Allele-Specific PCR

· For base change mutations (single nucleotide polymorphisms). · Use a primer whose 3' base matches the mutation. Will amplify one allele but not the other because the 3' end is not paired with the template in the wrong allele.

SSR Genetic Markers

. . Microsatellites (Simple Sequence Repeats: SSRs). Used for mapping the human genome -- the main marker system used today. · SSRs are short (2-5 bases) sequences that are repeated several times in tandem: TGTGTGTGTGTG is 6 tandem repeats of TG. . SSRs are found in and near many genes throughout the genome -- they are quite common and easy to find. . During normal replication of the DNA in the nucleus, DNA polymerase sometimes slips and creates extra copies or deletes a few copies of the repeat. . This happens rarely enough that most people inherit the same number of repeats that their parents had (i.e. SSRs are stable genetic markers), but often enough that numerous variant alleles exist in the population. . Mapping SSRs is a matter of having PCR primers that flank the repeat region, then examining the PCR products on an electrophoresis gel and counting the number of repeats. . SSRs are co-dominant markers: both alleles can be detected in a heterozygote. . If an SSR is a 3 base repeat within the coding region of a gene, it will create a tandem array of some amino acid. Certain genetic diseases, most notably Huntington's Disease, are caused by an increase in the number of repeats: once the number gets high enough the protein functions abnormally, causing neural degeneration. Such SSRs are called "tri-nucleotide repeats" or TNRs.

SSR Example

Cell-Based Molecular Cloning

· The original recombinant DNA technique: 1974 by Cohen and Boyer. · Several key players:

• 1. restriction enzymes. Cut DNA at specific sequences. e.g. EcoR1 cuts at GAATTC and BamH1 cuts at GGATCC. - Used by bacteria to destroy invading DNA: their own DNA has been modified (methylated) at the corresponding sequences by a methylase.
• 2. Plasmids: independently replicating DNA circles (only circles replicate in bacteria). Foreign DNA can be inserted into a plasmid and replicated. - Plasmids for cloning carry drug resistance genes that are used for selection. - Spread antibiotic resistance genes between bacterial species
• 3. DNA ligase. Attaches 2 pieces of DNA together.
• 4. transformation: DNA manipulated in vitro can be put back into the living cells by a simple process . - The transformed DNA replicates and expresses its genes.

Plasmid Vectors

· To replicate, a plasmid must be circular, and it must contain a replicon, a DNA sequence that DNA polymerase will bind to and initiate replication. Also called "ori" (origin of replication). Replicons are usually species-specific. – - Some replicons allow many copies of the plasmid in a cell, while others limit the copy number or one or two.

• Plasmid cloning vectors must also carry a selectable marker: drug resistance. Transformation is inefficient, so bacteria that aren't transformed must be killed.
• Most cloning vectors have a multiple cloning site, a short region of DNA containing many restriction sites close together (also called a polylinker) This allows many different restriction enzymes to be used.
• Most cloning vectors use a system for detecting the presence of a recombinant insert, usually the blue/ white beta-galactosidase system.

Basic Cloning Process

.Plasmid is cut open with a restriction enzyme that leaves an overhang: a sticky end .Foreign DNA is cut with the same enzyme. .The two DNAs are mixed. The sticky ends anneal together, and DNA ligase joins them into one recombinant molecule. .The recombinant plasmids are transformed into E. coli using heat plus calcium chloride. .Cells carrying the plasmid are selected by adding an antibiotic: the plasmid carries a gene for antibiotic resistance.

Protein Purification

Then the proteins can be removed from the bacteria by breaking the cell walls either chemically or by mechanical means. Protein solution OS by gel-electrophoresis Clone Addition of proteins and chemicals that dramatically enhance the production of a specific protein Genetic information which codes for protein of interest.