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Old 04-14-10, 03:58 PM   #11
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Plasmid isolation and purification:

The bacteria are grown to stationary phase, then a sample is used to make a plasmid minipreparation. First, centrifuge to pellet the cells and drain the supernatant. Add GTE buffer, which has EDTA that binds Mg2+ and stops DNA degradation. SDS/NaOH is then added. SDS is a detergent that dissolves the lipid components of the cell membrane and denatures proteins. The NaOH denatures chromosomal and plasmid DNA into single strands; the circular plasmid DNA remains linked. Adding KOAc neutralizes the miniprep and allows the DNA to renature. The large, chromosomal DNA strands though can't renature properly and become a tangled mess. The potassium acetate precipitates the SDS, proteins and lipids. Note that the DNA is in the supernatant, so after centrifugation, the supernatant is saved instead of the pellet this time. Next isopropanol is added, which selectively precipitates nucleic acids faster than proteins. The miniprep is centrifuged again, and the pellet is saved. It now contains DNA and RNA. Adding ethanol helps to wash away any remaining SDS and salts.
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Old 04-14-10, 04:10 PM   #12
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Restriction analysis of plasmid DNA:

The problem with miniprep DNA is that it contains impurities like fragments of DNA, RNA and DNases. Mg2+ ions are present as well, and allows DNase activity to continue. EDTA in the buffer chelates Mg2+ but since restriction enzymes require it as well, a balance needs to be reached between inhibiting DNase activity and facilitating restriction enzyme activity. RNA contaminants may also obscure results, so when purifying, RNases are added. These will be digested into very tiny fragments, and by virtue of their size, will run ahead of DNA fragments during electrophoresis.
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Old 04-14-10, 04:16 PM   #13
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Interpretation of electrophoresis results:

A background smear indicates that there is a lot of chromosomal DNA contamination

In the lane with presumably only uncut DNA, various bands may appear. The reasoning is that the DNA can take three forms: supercoiled, and because of it's compact size runs faster, nicked circles, which means the DNA is open and floppy and thus travels slowest, and linear, which travels intermediate to the other two.
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Old 04-14-10, 04:18 PM   #14
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If the DNA has "n" restriction sites, a linear strand will produce n+1 fragments, and circular one n-fragments.
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Old 04-14-10, 05:19 PM   #15
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Southern blot hybridization is a method used to identify a particular gene of interest, and requires a probe meant to hybridize to it. The probe is a synthetic oligonucleotide that has a tagged, ie: with fluorescence or radioactivity.

Run the DNA through electrophoresis. Treat the gel with acid to nick the DNA and make them linear, treat with a base to denature it into single strands, then add a buffer to neutralize the DNA.

Using a nitrocellulose sheet, blot the gel. Paper towels can help draw the DNA to stick to the sheet via capillary action. The sheet can be fixed by baking. The sheet is then incubated with the probe, which will bind to the gene of interest. To ensure stringency, the sheet is washed with a series of decreasing salt concentrations. Autoradiography etc. is then used to identify the probe.

Two variants of Southern blot hybridization are Northern blot to identify RNA using oligonucleotide probes, and Western blot to identify proteins using antibody probes.
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Old 04-14-10, 05:55 PM   #16
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Polymerase chain reaction (PCR) is a method used to make copies of a target sequence in vitro. The basic sequence of steps is denaturing, priming and extension. The invention of automated thermocyclers has made this technique more viable and produces results faster. The discovery of Taq polymerase from Thermus aquaticus, a more thermostable enzyme, retains it's function over higher temperatures and wider variances. However, it lacks proofreading capabilities, so after about 30 or so cycles, you lose fidelity. The number of molecules of the target sequence generated after "n" cycles is 2^n.

Two factors must be considered: specificity (how well primers target specific sequences) and fidelity (how accurately DNA is copied). Important considerations include:

- [enzyme] affects specificity and fidelity
- [dNTP] affects fidelity
- [Mg2+] affects specificity
- annealing temperature affects specificity
- type of enzyme affects fidelity
- number of cycles affects specificity
- primer design
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Old 04-14-10, 07:41 PM   #17
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PCR master mix must include Taq P, dNTP, primers, buffer. Nonspecific priming can begin with the addition of Mg2+.

Another consideration is to treat the sample with Chelex, which is a resin that binds metal cations under alkaline conditions.

Need two primers to bracket the target sequence: one is complementary to a sequence at the beginning of the target sequence (at 3' end of template), and another that is complementary to the sequence at the end of the target sequence on the antiparallel strand.

Degenerate primers can be reverse-engineered from proteins to produce primers that can anneal to multiple target sites.

Other important notes:

Because you need to plan out which primers to use, PCR requires some prior knowledge of the target sequence.

PCR is also highly sensitive to contaminants.

Because of the amplification capabilities, PCR can be used to detect for the presence of a target sequence from a very small sample, ie: from a crime scene. This is done by targeting short tandem repeats (STR), which vary between individuals in terms of length; polymorphism. The US FBI for example uses CODIS, a databank that stores DNA profiles.
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Old 04-14-10, 07:49 PM   #18
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Advanced PCR techniques:

RT-PCR - reverse transcription PCR, which uses cDNA as the template (derived from RNA using the enzyme reverse transcriptase). The advantage of this technique is that cDNA retains only the genes that are expressed; introns were removed during transcription of the RNA in the first place.

Real-time PCR (qPCR) - Same as regular PCR, except a third oligonucleotide is included in the PCR mix. This oligonucleotide consists of a fluorescent reporter, with two quenchers sandwiching it. In this state, no fluorescence is detected. It binds to the target site. As Taq P extends the target sequence, it cleaves the oligonucleotide nucleotides at a time. This results in one quencher being cleaved, revealing the reporter, which gets cleaved itself a bit later. This fluorescence can be detected and used to determine the number of target molecules are in the sample.
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Old 04-14-10, 07:58 PM   #19
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DNA Sequencing - determining the pattern of dNTP (ACGT) of a sequence.

Sanger Method - similar to PCR, except for having only one primer, the inclusion of fluorescent-labelled ddNTP, which bind like dNTP, except it's addition ends the extension. The idea is to make copies of every length possible. An automated sequencer draws the DNA up based on length (shortest to longest), and the fluorescence is detected by a laser. Each ddNTP emits a different color, thus indicating the sequence on the chromatogram readout. The sequence can then be analyzed via bioinformatics.

Note: Pyrosequencing is the new, upcoming method for DNA sequencing.
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Old 04-14-10, 08:12 PM   #20
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Bioinformatics is a discipline that uses databases and searches to support molecular biology and genetics.

For our purposes, we use US National Institute of Health's (NIH) genetic database, the National Center for Biotechnology Information (NCBI). We used two tools: BLAST (Basic local alignment search tool), and ClustalW2. The one we didn't use was Entrez, which lets you search but also interfaces with other databases, and not just the NCBI's.

BLAST takes your sequence and compares it with all other sequences in it's database, and produces a list of possible matches, kind of like searching on Google. You can input nucleotide sequences or protein sequences, and can search different databases, ie: human, mouse, etc.

ClustalW2 on the other hand takes the several sequences you input, and compares them against each other. It then produces a multiple sequence alignment or a phylogenetic tree.
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