Chapter 5 - Molecular Tools for Studying Genes and Gene Activity

Automated DNA Sequencing

Manual sequencing is powerful but slow. Instead this technique uses ddNTPs tagged with different fluorescent molecules. The products of each ddNTP will fluoresce a different color. Four reactions are completed, then mixed together and run out on one lane of a gel.

DNA Mapping

Mapping occurs via the identification of DNA landmarks.

Mapping and Quantifying Transcripts

Mapping refers to locating the start and end. Quantifying refers to the amount of transcript existing at one time.

Sanger Method of DNA Sequencing

A primer that is complementary to the upstream portion of the DNA segment you want to sequence is allowed to anneal to the DNA strand. In four separate test tubes, DNA elongation from the primer is allowed to occur with both dNTPs and one type of ddNTP per test tube. The products are then electrophoresed and are analyzed.

Labeled/Radioactive Tracers

Allow very small quantities of substances to be detected and quantifiable.

Run-Off Transcription

Assay used to measure the rate of in vitro transcription. DNA fragment containing gene to transcribe is cut with restriction enzyme in middle of transcription region. Transcribe the truncated fragment in vitro using labeled nucleotides, as polymerase reahces truncation it "runs off" the end. Measure length of run-off transcript compared to location of restriction site at 3' end of truncated gene.

Site-Directed Mutagenesis

Cloned genes permit biochemical microsurgery on proteins. Specific bases in a gene may be changed. Amino acids at specific sites in the protein product may be altered as a result. Effects of those changes on protein function can be observed.

Gel Filtration Chromatography

Column chromatography that is used to separate out by size. The columns are filled with porous resins. The pores of the resins are specifically sized in order to allow the substance of interest to fit inside. Therefore, smaller substances are caught up inside the porous resins, whereas larger ones are eluted first.

Affinity Chromatography

Column chromatography that is used to separate out substances that have a specific affinity for something. Also uses resins, but the resin used contains a substance to which the molecule of interest has a strong and specific affinity for. The sample is loaded into the column and fractions are allowed to elute out. Only the molecule of interest will be bound to the resins with their specific affinity substance. The molecule of interest is eluted last from the column using a specific solution that is able to disrupt their specific binding. This technique is especially effective in separating proteins via immunoglobulins. It also offers the highest "resolution" of separation compared to the previous column chromatography techniques.

Southern Blots

DNA gel electrophoresis is run. The separated strands of DNA are transferred to some filter (nitrocellulose) by diffusion; DNa can also be electrophoresed onto the filter. The filter is then treated with alkali to denature the DNA, resulting ssDNA binds to the filter. A labeled cDNA probe that is complementary to the DNA strand of interest is the applied to the filter. A positive band should be detectable where hybridization between the probe and DNA occurred. The probe hybridizes and a band is generated corresponding to the DNA fragment of interest. The bands are visualized via x-ray autobiography.

How DNA Gel Electrophoresis Works

DNA is has an overall negative charge due to the phosphates of its backbone structure. Thus when an electric current is applied to the gel system, the DNA will be pulled through the gel along with the electric current. DNA moves according to its size. The smaller the DNA fragment, the more mobile it is; the larger the DNA, the more impeded its motion through the gel is. The DNA is stained with a fluorescent dye (ethidium bromide) that intercalates between the bases.

Restriction Map

DNA mapping where the identifying DNA landmarks are restriction sites.

PCR-based Site-Directed Mutagensis

Denature dsDNA and hybridize to one of the strands a mutagenic primer. Allow replication of new strand with mutagenic primer. Conduct PCR.

Nucleic Acid Hybridization

Hybridization is the ability of one single stranded nucleic acid to form a double helix with another single strand of complementary base sequence.

In Situ Hybridization

Labeled probes can be used to hybridize to chromosomes and reveal which chromosome contains the gene of interest. The chromosomes from a cell are spread. The DNA is partially denatured, creating single stranded regions, allowing hybridization to occur with a labeled probe. The chromosomes can then be stained and detected.

How to do DNA Gel Electrophoresis

Melt agarose solution and pour it into a mold with a removable comb. Once cooled, the hardened agarose gel is transferred to a device that will send an electrical current from one end of the gel to the other. DNA samples are then loaded into the slots of the gel formed by the teeth of the comb; a DNA marker/ruler is usually utilized and loaded into the first slot. The electrophoresis machine is then turned on, allowing an electric current to flow from the anode to the cathode.

High Throughput Sequencing

Once an organisms's genome sequence is known, very rapid sequencing techniques can be applied to sequence the genome of another member of the same species. It produces relatively short reads or contiguous sequences (25-30 bp OR 200-300 bp, depending on the method used) that can easily be pieced together if a reference sequence is available.

DNA Fingerprinting

Process is a Southern blot. Cut the DNA under study with restriction enzyme; ideally, the cut is made on either side of the minisatellite and NOT within. Digested DNA is run on a gel and then blotted via southern blot. The probe utilized is labeled, allowing ease of visualization. Parts of the pattern of a DNA fingerprint are inherited in a Mendelian fashion. This can be used to establish parentage (or relation). Has the potential to identify criminals in a court of law and subsequently disprove someone of crimes.

Liquid Scintillation Counting

Radioactive emissions from a sample create photons of visible light are detected by a photomultiplier tube in the process of liquid scintillation counting. First, the radioactive band form the gel is removed to a vial containing scintillation fluid. The scintillation fluid contains a fluor that fluoresces when hit with radioactive emissions. It acts to convert invisible radioactivity into visible light.

Detecting Nucleic Acids With a Nonradioactive Probe

Replication occurs with biotinylated dUTP (biotin molecule attached to backbone of uracil bases). The double strand is denatured and is then allowed to hybridize to DNA strand of interest. Avidin-alkaline phosphatase is mixed in with the hybrid double strand (the avidin will bind to the biotin). After allowed to bind, a phosphorylated substance is mixed into the solution and is allowed to bind to the avidin via dephosphorylation. Cleavage of the substance off of the avidin phosphatase will produce a chemiluminscent product which can then be detected with an x-ray film.

Fluorescence in situ hybridization (FISH)

Same procedures as a normal in situ hybrid., but the probe being used can be detected with a fluorescent antibody.

Two-Dimensional Gel Electrophoresis

Samples are run in 2 gels. The first dimension separates using one concentration of polyacrylamide at one pH. Second dimension uses different concentration of polyacrylamide and pH. Proteins move at different pH values without SDS and at different acrylamide concentrations.

DNA Sequencing

Sanger, Maxim, and Gilbert developed 2 methods for determining the exact base sequence of a cloned piece of DNA. Modern DNA sequencing is based on the Sanger method and uses dideoxy nucleotides to terminate DNA synthesis. This process yields a series of DNA fragments whose size is measured by electrophoresis. The last base in each fragment is known as that dideoxy. Ordering of the fragments by size tells the base sequence of the DNA.

Immunoblot/Western Blot

Similar concepts to Southerns. Start by electrophoresing the proteins (SDS-PAGE or 2D). The separated proteins are then blotted from the gel to a membrane. "Hybridization" occurs via the binding of an antibody/antiserum to target protein. Addition of a second labeled antibody is used to bind to the first antibody for visualization. The first antibody is very specific whereas the second antibody is very general.

DNA Fingerprinting and DNA Typing

Southern blots are used in forensic labs to identify individuals from DNA-containing materials. Minisatellite DNA (discovered by Alec Jeffrys) is a sequence of bases repeated several times. Individuals differ in the patter of repeats of the basic sequence. The difference is large enough that 2 people have only a remote change of having exactly the same pattern of repeats.

Pulsed-Field Gle Electrophoresis (PFGE)

Special techniques are required for DNA fragments larger than about 1 kb. Instead of a constant current being applied, an alternating long pulses of current in the forward direction (from anode to cathode) followed by shorter pulses of current in an opposite/sideways direction.

Illumina Company High Throughput Sequencing

Starts by attaching short pieces of DNA to a solid surface, amplifying each DNA in a tiny patch on the surface, then sequencing the patches together by extending them one nucleotide at a time using fluorescent chain-terminating nucleotides, whose fluoresce reveals their identity.

Phosphorimaging

Technique that is more accurate in quantifying amount of radioactivity in a substance. The response to radioactivity is more linear. Instead of placing the gel with radiolabeled DNA onto an X-ray film, the gel is placed in contact with a phosphorimager plate. The plate then absorbs electrons (from beta emissions) that excite molecules on the phosphorimager plate; plate will remain in an excited state until the plate is scanned.

Nonradioacitve Tracers

Techniques that maintain the sensitivity of radioactive tracers, but do not come with the associated dangers: health exposure, handling, and disposal. The increased sensitivity to these techniques comes from a multiplier effect of an enzyme that is coupled to a probe for molecule of interest.

Autoradiography Analysis

The developed film can be qualitatively assessed for the relative quantity of DNA. Larger, thicker black bands on X-ray film means more DNA. Quantitatively however, a densitometer can be used. Uses light absorbance. The darker the band, the more light is absorbed.

Autoradiography

The means of detecting radioactive compounds with a photographic emulsion. An X-Ray film can be used as the photographic emulsion. DNA is separated on a gel and is radiolabeled. The gel is placed in contact with the film for hours/days. Radioactive emissions from the labeled DNA expose the film causing dark bands to show after film development.

2D Sodium Dodecyl Sulfate Polyacrylamid Gel Electrophoresis (2D SDS-PAGE)

Two process method: 1. Use isoelectric focusing gel: mixture of proteins are electrophoresed through a gel containing a pH gradient. Proteins move according to their isolelectric point (where the overall charge on the protein molecule is zero). Tube gel is removed and ready to be used in the second step. 2. SDS-PAGE: tube gel sample is placed at the top of a standard polyacrylamide gel. Proteins are only partially resolved by their isoelectric points. This will resolve by protein size. Same concept as DNA GE, smaller proteins are more mobile than larger ones.

Pyrosequencing

Type of high throughput sequencing. An automated system with the advantages of speed and accuracy. The nucleotides are added one by one and the incorporation of a nucleotide is detected by a release of pyrophosphate, which leads to a flash of light which can then be detected.

S1 Mapping

Use S1 mapping to locate the ends of RNAs and to determine the amount of a given RNA in cells at a given time. Label an ssDNA probe that can only hybridize to transcript of interest. Probe must span the sequence start to finish. After hybridization, treat with S1 nuclease which degrades ssDNA and RNA. Transcript protects part of the probe from degradation. Size of protected area can be measured by gel electrophoresis.

Northern blots

Used to identify and detect RNA strands. RNA is obtained from different tissues. RNA is run on an agarose gel and is blotted to a membrane. It is then hybridized to a labeled cDNA probe. This method can be used to tell us how actively is the corresponding gene expressed in different tissues. We can then quantify using a densitometer.

Gel Electrophoresis

Used to separate out different species of Nucleic Acid (typically DNA) and protein. It mainly separates out these molecules based off of size and charge.

Ion-Exchange Chromatography

Uses a resin to separate substances by their charge. The resin is placed inside a column and the sample is loaded onto the column material. The resin can either be positively charged (attracting negatively charged substances and allowing positively charged ones to elute first) or negatively charged (attracting positively charged substances and allowing negatively charged ones to elute first). This is specifically useful for separating out proteins.

Primer Extension Schemtaic

Works to determine the 5' end of a transcript to one-nucleotide accuracy. Start with in vivo transcription, harvest cellular RNA containing desired transcript. Hybridize labeled oligonucleotide primer. Reverse transcriptase extends the primer to the 5' end of transcript. Denature the RNA-DNA hybrid and run the mix on a high resolution DNA gel. You can estimate transcript concentration also.