Thin-Layer Chromatography Is an Important Technique for Lipid Analysis Lipids can be isolated, separated, and studied using nonpolar solvents such as acetone and chloroform Thin-layer chromatographyis used to separate different kinds of lipids based on their relative polarities A glass plate is coated with silicic acid and lipids are spotted onto a position near the bottom of the plate called the origin
Principle of separation of lipids via TLC A nonpolar organic solvent moves up the plate by capillary action, taking different lipids with it to varying degrees Nonpolar lipids have little affinity for the silicic acid on the plate, and so move readily with the solvent, near the solvent front Polar lipids will interact variably (depending on how polar they are) with the silicic acid, and their movement will be slowed proportionately
The Lipid Bilayer Is Fluid The lipid bilayer behaves as a fluid that permits the movement of both lipids and proteins Lipids can move as much as several mm per second within the monolayer Lateral diffusion can be demonstrated using fluorescence recovery after photobleaching(FRAP)
Measuring lipid mobility with FRAP Investigators label lipid molecules in a membrane with a fluorescence dye A laser beam is used to bleach the dye in a small area, creating a dark spot on the membrane The membrane is observed afterward to determine how long it takes for the dark spot to disappear, a measure of how quickly new fluorescent lipids move in
The Membrane Consists of a Mosaic of Proteins: Evidence from Freeze-Fracture Microscopy Support for the fluid mosaic model came from studies involvingfreeze-fracturing A bilayer or membrane is frozen and then hit sharply with a diamond knife The resulting fracture often follows the plane between the two layers of membrane lipid
Freeze-fracture analysis of membranes When a fracture plane splits a membrane into its two layers, particles the size and shape of globular proteins can be seen The E surface is the exoplasmic face and the P surface is the protoplasmic face Artificial bilayers without added protein show no particles
Gel Electrophoresis Electrophoresis is a group of techniques that use an electric field to separate charged molecules How quickly a molecule moves during electrophoresis depends on both charge and size Electrophoresis uses various support media most commonly polyacrylamide or agarose
Agarose Gel electrophoresis • Positive electrode (anode) • Negative electrode (cathode) • One electrode is applied, charged molecules in the samples migrate through pores of the gel toward their pole of attraction. • Mobility is also dependent on size and shape. Smaller molecules maneuver more easily through pores • Used to determine mutations in DNA, new genes, restriction sites in DNA, etc.
Southern Blotting • Transfer of DNA to a nitrocellulose filter • Used to identify DNA sequences using a DNA or RNA probe
Electrophoresis of membrane proteins Membrane fragments are solubilized in SDS, which disrupts protein-protein and protein-lipid associations The proteins are thus coated with negatively charged detergent molecules The proteins are loaded onto a polyacrylamidegel and an electric potential applied
Electrophoresis of membrane proteins (continued) The negatively charged proteins run toward the positively charged bottom of the gel Polypeptides move through the gel with the smallest moving fastest The gel is stopped when the smallest proteins reach the bottom, and is stained with a dye such as Coomassie brilliant blue to show the proteins
Additional techniques using electrophoresis Two-dimensional SDS - PAGE (polyacrylamide gel electrophoresis) separates proteins in two dimensions, first by charge and then by size Following electrophoresis, polypeptides can be identified by Western blotting In this technique proteins are transferred to a membrane and bound by specific antibodies
PCR cycle • Each cycle consist of three stages: • Denaturation • Reaction tube is heated to ~94-960C causing separation of the target DNA into single strands • Hybridization (Annealing) • Tube is cooled slightly to ~60-650C, which allows the primers to hydrogen bond to complementary bases at opposite ends of the target sequences • Extension (Elongation) • Temperature is raised slightly to ~70-75°C and DNA polymerase copies the target DNA by binding to the 3`end of each primer. • At the end of one cycle- target DNA has doubled – usually run about 30-40 cycles. • http://www.dnalc.org/ddnalc/resources/pcr.html
Type of DNA polymerase important • Must use an enzyme that is suitable for the various temperature changes. • Most popular is Taq DNA polymerase. Isolated from archaea called Thermus aquaticus, a species that is adjusted to hot temperature. • Named the molecule of the year by the Journal Science in 1989.
Advantage of PCR • Amplify millions of copies of target DNA from a very small amount. • After 20 cycles approximately 1 million copies are produces -2 20 • New Applications of PCR • Quantitative Real-time PCR –used primers with fluorescent dyes to quantify amplification reactions as they occur
Cloning PCR products • PCR is often used instead of library screening • Disadvantage of PCR cloning is that you need to know something about the DNA sequences to design primers • Use PCR to clone gene. • Gene is amplified using primers
3.4 What Can You Do with a Cloned Gene? Applications of Recombinant DNA Technology • DNA Sequencing • Important to determine the sequence of nucleotides of the cloned gene • Reasons for knowing the DNA sequence: • Deduce the amino acid sequence of a protein encoded by a cloned gene • Determine the exact structure of a gene • Identify the regulatory elements such as promoter sequences • Identify differences in genes created by gene splicing • Identify genetic mutations
DNA sequencing Common Methods are: PCR sequencing and Computer-automated DNA sequencing • Most widely used sequencing method developed in 1977 by Frederick Sanger • Chain termination sequencing (Sanger method)
DNA Sequencing Technique • A DNA primer is hybridized to denatured template DNA (plasmid-containing cloned DNA) • This is added to a reaction tube containing • Deoxyribonucleotides and DNA polymerase • A small amount of a modified nucleotide called a dideoxyribonucleotide (ddNTP) is mixed in with the target DNA, primer, polymerase, and deoxyribonucleotides.
Dideoxynucleotide Procedure for DNA sequencing • What is a dideoxynucleotide? • Human-made molecule • Lacks a hydroxyl group at both the 2’ and 3’ carbons of the sugar moiety (normal deoxyribonucleotide has a 3’OH group)
DNA replication • Recall normal DNA replication • Nucleoside triphosphate is linked by it 5’alpha phosphate group to the 3’hydroxyl group of the last nucleotide growing chain. • If dideoxynucleotide is incorporated at the end of the growing chain, DNA synthesis stops because a phosphodiester bond cannot be formed with the next incoming nucleotide.
Steps involved • Anneal a synthetic oligonucleotide (17-24mer) to a predetermined segment of a strand of the cloning vector near the insertion site of the cloned DNA (radioactively labeled primer) • This acts as a primer sequence by providing a 3’ hydroxyl group for initiation of DNA synthesis
continued • The primed DNA sample is partitioned into four separate tubes. Each tube contains four deoxyribonucleoties, DNA polymerase, cloned DNA to be sequenced, a one modified dideoxyribonucleotide. • Recall chain growth stops as soon as a dideoxynucleotide is incorporated. (at the 3’terminus)
Sequencing continued • After DNA synthesis, each reaction tube will contain unique oligonucleotide. • DNA molecules are separated by polyacrylamide gel electrophoresis ( good for small sizes up to a single nucleotide) • Autoradiograph shows the radiolabeled DNA fragments that were produced during the enzymatic DNA synthesis step. • Each of the four lanes on the autoradiograph corresponds to a reaction tube that contained one of the four dideoxynuclotides.
How is it read? • As accurately as possible, the order of the bands are read from the bottom to the top of the autoradiograph (the radiolabeled fragment closes to the bottom) • Remember you are reading the complementary strand to the template strand • Normally can resolve up to 350 bands
Limitation of DNA sequencing • Used to sequence approximately 200- 400 nucleotides in a single reaction • Longer than 400 must run multiple reactions to create overlapping sequencing • Piece together to determine the entire sequence • Cumbersome for large-scale sequencing like Human Genome Project
Automated DNA Sequencing • Minimizes manual manipulations • Dideoxynucleotide method forms the basis of automated DNA sequencing • Highly accurate can resolve 20,000 bases per hour • Sequence analysis carried out with four different fluorescent dyes (for each dideoxy) • Samples are still separated with polyacrylamide gel or polymer-filled capillary tube. • Fluorescent dye emits a narrow spectrum of light. The fluorescent signals are read by a computer and converted to a sequence of nucleotides.
Computer automated sequencing • ddNTP’s are each labeled with a different fluorescent dye • Samples are separated on a single-lane capillary gel that is scanned with a laser beam • Creates different color patterns for each nucleotide • Converted by computer to the sequence
3.4 What Can You Do with a Cloned Gene? Applications of Recombinant DNA Technology • Chromosome Location and Copy Number • Identify the chromosome location of the cloned gene • Determine if the gene is present as a single copy in the genome • Fluorescence in situ hybridization (FISH) • Chromosomes are isolated from cells and spread out on glass slide • cDNA probe for gene of interest is labeled with fluorescent nucleotides and incubated with slides
Fluorescence in situ hybridization (FISH) • Identify which chromosomes contains a gene of interest. • continued • Probe will hybridize with complementary sequence on the slide. • Fluorescently labeled probe is illuminated to indicate the presence of that gene • Align chromosomes (karotype) to determine which one. • Usually for multiple copies of genes, genetic disorders (fetal disorders- Downs syndrome)