Techniques

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 chromatography is 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

Figure 7-9

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

Figure 7-11

The Membrane Consists of a Mosaic of Proteins: Evidence from Freeze-Fracture Microscopy

• Support for the fluid mosaic model came from studies involving freeze-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

Figure 7-16A

Figure 7-16B

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 electrophoresisPositive 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 polyacrylamide gel 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

Figure 7-22

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)

3.4 What Can You Do with a Cloned Gene? Applications of Recombinant DNA Technology

• Chromosome Location and Copy Number– Southern blotting- Developed by Ed Southern in 1975

• Digest chromosomal DNA into small fragments with restriction enzymes

• Fragments are separated by agarose gel electrophoresis• Gel is treated with alkaline solution to denature the DNA• Fragments are transferred onto a nylon or nitrocellulose

filter (called blotting)• Filter (blot) is incubated with a probe and exposed to film

by autoradiography• Number of bands on film represents gene copy number

Southern Blotting and PCR

• http://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120078/bio_g.swf::Southern+Blot

• http://www.sinauer.com/cooper/4e/animations0411.html

• http://www.maxanim.com/genetics/PCR/PCR.htm

Setting up the gel sandwich

• Gel is placed under the nylon or nitrocellulose membrane, filter paper, paper towels, and weight allowed for wicking the salt solution through gel

• DNA is transferred from the gel to the filter by capillary action or by current

• Blot is then incubated with nonradioactive or radioactive probe.

mRNA analysis

• All cells in the body (except germ cells) carry the same set of genes, but only a subset of those genes is active in a particular cell or tissue.

• You can determine which genes are being transcribed into mRNA (expressed).

• One a gene is cloned out of a library or you make a probe it can be used in the analysis of mRNA

3.4 What Can You Do with a Cloned Gene? Applications of Recombinant DNA Technology

• Studying Gene Expression– Techniques involve analyzing mRNA produced by a tissue– Northern blot analysis• Basic method is similar to Southern blotting• RNA is isolated from a tissue of interest, separated by

gel electrophoresis, blotted onto a membrane, and hybridized to a probe. • Amount of RNA from different sites, tissues, exposures

compared.

Northern Blot

Problems with Northerns

• Not particularly sensitive and may not detect a gene transcribed at a low level.

• Northern blots require a fairly large quantity of mRNA, which may be difficult to obtain from a small tissue, such as the embryonic kidney

RT-PCR

– Sometimes the amount of RNA produced by a tissue is below the level of detection by Northern blot analysis

– What can we use to amplify genes?– Can PCR amplify RNA?– Reverse transcription PCR• Enzyme reverse transcriptase• Reverse transcription of mRNA is performed –

converted into double-stranded cDNA• cDNA is then amplified with a set of primers specific

for the gene of interest• Products electrophoresed on agarose gel

Real time PCR

• Used to determine the amount of PCR product during an experiment

• Quantify amplification reactions as they occur in “real time” or quantitative PCR (qPCR)

• Basic procedure uses a specialized PCR (thermal cycler) machine– Laser to scan the PCR tube– Reaction contains a dye-containing probe or DNA binding

dye that emits fluorescent light when illuminated by the laser.

– Light emitted correlates to amount of PCR product amplified

Two approaches to Real-time PCR

• SYBR Green– Dye that binds double-stranded DNA– As more dsDNA is copied, more DNA binds to SYBR Green– Increases amount of fluorescent light emitted

• Taqman– Probes complementary to specific regions of target DNA between the

primers– Two dyes: reporter dye at the 5’end of probe and can release

flurorescent light, the other dye is called a quencher, attached to the 3’ end of the probe

3.4 What Can You Do with a Cloned Gene? Applications of Recombinant DNA Technology

• Studying Gene Expression– In situ hybridization

• Used to determine the cell type that is expressing the mRNA

• Tissue of interest is preserved in a fixative solution and embedded in a wax-like substance

• Tissue can be sliced into very thin sections attached to microscope slides

• Slides are incubated with a probe to the gene of interest• Probe hybridizes with mRNA in cells• Probe is detected

In Situ Hybridization

• Means “in place” hybridization is a more sensitive method for visualizing gene activity directly in fixed cells or tissues.

• Rather than extracting mRNA from cells, the mRNA is left in place and the entire cell, tissue, or embryo is fixed using paraformaldehyde (prevents breakdown of molecules)

Procedure for in situ

• Early procedure – cut tissue into very thin sections, adhere to microscope slides. Use a probe to look for mRNA directly in tissue.

• Disadvantages – safety, time consuming (preparing slides), and difficulty in observing the gene expression in the overall tissue.

DNA Microarrays

• How do these genes work together?• Different cells express different sets of genes

at different levels• DNA array is a means to analyze the

expression patterns of thousands of genes at a time

• Study all the genes expressed in a tissue quickly

3.4 What Can You Do with a Cloned Gene? Applications of Recombinant DNA Technology

• Studying Gene Expression– Gene microarrays• DNA microarray analysis• Single-stranded DNA molecules are attached onto a

slide using a robotic arrayer fitted with tiny pins

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DNA or Gene Microarrays• A microarray, also known as gene chip, is created using a

small glass microscope slide.• Single-stranded DNA molecules are attached to the slide

using a computer-controlled high-speed robotic arm called an arrayer, fitted with a number of tiny pins.

• Each pin contains millions of copies of different DNA molecules (cDNA’s from different genes).

• DNA is fixed to the slide at specific locations that are recorded by the computer. (one microarray can have over 10,000 spots of DNA).

• How do we use the microarray?

How do you use a microarray?• http://www.dnalc.org/ddnalc/resources/dnaarray.html

• Extract mRNA (cDNA) from a tissue of interest• The mRNA is then tagged with a fluorescent dye and

incubated overnight with the microarray.• mRNA hybridize to spots on the microarray that contain

complementary DNA sequences.• Microarray is washed and scanned by a laser that cause the

mRNA hybridized to the microarray to fluoresce. • Tell you which genes are expressed in your sample and how

much.

Data analysis

• The fluorescent spots reveal which genes are expressed in the tissue of interest

• Intensity of fluorescence indicates that relative amount of expression

• Brighter spot – more mRNA expressed in that tissue

• Microarrays can also be run by labeling cDNAs from two or more tissues with different colored fluorescent dyes.

3.4 What Can You Do with a Cloned Gene? Applications of Recombinant DNA Technology

Uses of Microarray analysis

• Purchase an entire genome on a microarray- humans, yeast and bacteria.

• Compare pattern of expressed genes under different conditions (stress, inflammation, disease, etc).

• Cancer cells can be compared to normal cells to look for genes that may be involved in cancer formation. Develop new drug therapy strategies.

RNA interference

• 1998, Mello and Fire used double-stranded pieces of RNA (dsRNA) to inhibit or silence expression of genes in the nematode roundworm

• Technique is called RNA interference or RNAi– ssRNA combines to mRNA– Degrade mRNA or block translation of RNA

• http://www.wiley.com/college/pratt/0471393878/student/animations/dna_sequencing/index.html

• http://www.bio.davidson.edu/courses/genomics/chip/chip.html