Manipulatingnucleicacids 111109075650-phpapp02

Mrs. Ofelia Solano Saludar

Department of Natural Sciences University of St. La Salle

MANIPULATING MOLECULES IN LIVING CELLS:

NUCLEIC ACIDS

Hybridization techniques allow identification of specific macromolecules, e.g. proteins, and DNA or RNA sequences based on ability of NA to bind specifically to labeled, known, single-stranded NA sequences.

Separation methods employed range from enzyme digestion, chromatography, electrophoresis or centrifugation.

Using restriction endonucleases which cleave DNA at specific sequences, recombinant DNA and other facets of biotechnology are benefited by these methods.

Wait until next semester for this…

Biochemical and Molecular Methods

Nucleic Acid Hybridization. (A) If the DNA helix is separated into 2 strands, the strands should reanneal, given the appropriate ionic

conditions and time. (B) If DNA is separated into its 2 strands, RNA should be able to bind to the genes that encode it. If present in

sufficiently large amounts compared with the DNA, the RNA will replace one of the DNA strands in this region.

Combined with reporter molecules, hybridization enables cytogeneticists to create probes to detect, quantify, visualize

DNA/RNA location, and monitor their activity in a given cell/ tissue of interest.

Construction of a human genomic DNA library. A genomic library is usually stored as a set of bacteria, each bacterium carrying a

different fragment of human DNA. The entire collection of

clones derived from one mRNA preparation

constitutes a cDNA library. Because the cells of different tissues produce distinct sets

of mRNA molecules, a distinct cDNA library is obtained for

each type of cell used to prepare the library.

http://highered.mcgraw-hill.com/olc/dl/120078/bio_h.swf

The synthesis of cDNA.Total mRNA is extracted from

a particular tissue. The enzyme reverse transcriptase produces DNA copies (cDNA)

of the mRNA molecules. A short complement to the poly-

A tail at the 3' end of the mRNA acts as a primer for the reverse transcriptase, which then copies the RNA into a complementary DNA chain, thereby forming a DNA/RNA

hybrid helix. Treatingthe DNA/RNA hybrid with RNaseH creates nicks and

gapsin the RNA. DNA polymerase used to synthesize the 2nd DNA strand synthesize the

bound RNA molecule, resulting to sequences at the 5‘ end to

be absent from cDNA libraries.

Gene A is infrequently transcribed compared to gene B. In the genomic DNA library, both the

introns (green) and the nontranscribed DNA

(pink) are included in the clones. Most clones

contain only part of the coding sequence of a

gene (red). In the cDNA clones, the intron

sequences (yellow) have been removed by RNA

splicing during the formation of the mRNA (blue). Because gene B

is transcribed more frequently than gene A in the cells from which the cDNA library was

made, it is represented much more frequently

than A in the cDNA library. In contrast, A and

B are in principle represented equally in

the genomic DNA library.

The differences between cDNA clones and genomic DNA clones derived from the same region of

DNA.

Preparation of a bacteriophage λ cDNA library. A mixture of mRNAs is isolated and used to produce cDNAs

corresponding to all the cellular mRNAs (1–3). These single-stranded cDNAs (light green) are then converted into double-stranded cDNAs, which are

treated with EcoRI methylase to prevent subsequent digestion by EcoRI (4 –6 ). The protected double-stranded cDNAs

are ligated to a synthetic double-stranded EcoRI-site linker at both ends

and then cleaved with the corresponding restriction enzyme, yielding cDNAs with

sticky ends (red letters); these are incorporated into λ phage cloning

vectors, and the resulting recombinant λ virions are plated on a lawn of E. coli

cells (7–9 )http://highered.mcgraw-hill.com/olc/dl/120078/micro10.swf

Phage cDNA libraries canbe screened with a

radiolabeled probe to identify a clone of

interest. The appearance of a spot on the

autoradiogram indicates the presence of a recombinant clone containing DNA complementary to the probe. The position of the spot on the autoradiogram is themirror image of the position on the original petri dish of thatparticular clone. Aligning the

autoradiogram with the original petri dish will locate the

corresponding clone from which infectious phage particles can be

recovered and replated at low density, resulting in well-

separated plaques. Pure isolates eventually are obtained by repeating the hybridization

assay.

(A) PCR primers that flank the stretch of DNA to be cloned

are added to purified chromosomal DNA, and many PCR cycles of are completed. Since only the DNA between the primers is amplified, PCR provides a way to obtain a

short stretch of chromosomal DNA selectively in a virtually pure form. (B) To use PCR to

obtain a cDNA clone of a gene, mRNA is first purified

from cells. The first primer is then added to the population

of mRNAs, and reverse transcriptase is used to make a complementary DNA strand. The 2nd primer is added, and

the single-stranded cDNA molecule is amplified through

many PCR cycles. For both types of cloning, the

nucleotide sequence of at least part of the region to be

cloned must be known beforehand.

Use of PCR to obtain a genomic or cDNA clone.

Cloning genomic DNA by the PCR technique. Each cycle of the reaction begins with a brief heat treatment to separate the two

strands (aka, heat cycler). Hybridization to complementary sequences in the two DNA strands take place to produce 4 dsDNA

molecules, and the 5-min. cycle is repeated 20-30x to produce amplified DNA copies. Trace amounts of RNA can be analyzed in the

same way by first transcribing them into DNA with reverse transcriptase.

A PCR reaction using 2 primers that bracket a particular microsatellite, or VNTR sequence, produces a different

pair of DNA bands from each individual. Each band represents VNTR sequences inherited from the mother

and father. Although some individuals have several bands in common, the overall pattern is quite distinctive

for each.

..\..\..\genetics\videos\pcr.exe

http://highered.mcgraw-hill.com/olc/dl/120078/micro15.swf

.

The PCR cloning technique has

largely replaced Southern

blotting for the diagnosis of

genetic diseases and for the

detection of low levels of viral

infection.

Bands obtained from a set of PCR reactions

which amplifies different VNTR sequences, can

serve as a "fingerprint" to identify each

individual. The starting material for the PCR

reaction can be a single hair or blood left at the

crime scene.http://highered.mcgraw-hill.com/olc/dl/120078/bio20.swf

Methods for mRNA

Isolate populations of mRNA that characterize certain cell types and are absent in all others

Determine the temporal and spatial locations of RNA expression

1.Northern blot- extract total mRNA from the specimen and separate it by electrophoresis.

Bands produced are complementary mRNA to the probe, and intensity of bands are proportional to amount of specific mRNA.

Northern blotting.

2.Ribonuclease protection

Visualized radioactivity reveals a range of intensity proportional to the

content of specific RNA in the sample.

The mixture is separated on a sequencing gel

Hybrids are digested with ribonucleases (double-stranded ones will be protected

from digestion)

32P labeled probe is hybridized in solution with total RNA from

specimen

3.Reverse-transcription polymerase chain reaction (RT-PCR)

The intensity of bands bears some relation to the initial amount of mRNA in the

sample.

The reaction mixture is run on a gel and the DNA bands are visualized in the usual way.

The sequence between the primers is amplified by repeated cycles of synthesis, melting and

hybridization.

Two oligonucleotides are added, chosen to correspond to sequences in the target

cDNA.

Total RNA from sample is reverse-transcribed into cDNA using reverse transcriptase.

Methods for DNA

1.In situ hybridization- designed to reveal the spatial domains of gene expression in a specimen.

An antisense probe is synthesized in vitro complementary to the mRNA to be detected. This is hybridized to the specimen and then visualized.

Probes usually include extra chemical groups recognizable by a commercially available antibody for detection (e.g. digoxigenein or DIG, a plant sterol)

Radioactive probes are used in radioactive in situ hybridization (RISH), fluorescent dyes are used in FISH.

Fluorescence In Situ Hybridization (FISH) begins with a DNA probe and a target sequence. The DNA probe is labeled by indirect labeling (left) and

direct labeling (right). The labeled

probe and the target DNA are

denatured to yield single stranded

DNA. They are then combined, which

allows the annealing of

complementary DNA sequences.

In situ hybridization performed on a whole chick embryo that have been fixed without being

sectioned. The probe used recognizes the mRNA encoding Pax6 in the chick embryo. This probe is labeled not with a radioactive isotope, but with a modified UTP. To create this probe, a

region of the cloned Pax6 gene was transcribed into mRNA, containing UTP conjugated with digoxigenin. This does not interfere with the coding properties of the resulting mRNA, but does make it recognizably different from any

other RNA in the cell.

2.Southern blot- to evaluate DNA extracts from tissue samples.

This is a type of nucleic acid hybridization test in which single-stranded DNA from two sources interact.

Strands with similar nucleic acid sequences will anneal by base pairing (A with T, and G with C) to form double-stranded molecules.

One of the single-stranded DNA molecules is a unique portion of the gene of interest, and is radioactively labeled so it can be detected on photographic film (the probe).

http://highered.mcgraw-hill.com/olc/dl/120078/bio_g.swf

Southern blotting. (1) DNA is treated with restriction enzymes, and the resulting restriction fragments of

DNA are placed in a gel. (2) After the fragments of DNA are separated on the gel by electrophoresis, the DNA

is denatured into single strands. (3) The gel is then placed on a support on top of a filter paper saturated with high-ionic-strength buffer. Nitrocellulose paper or

a nylon filter is placed on the gel, and towels are placed atop the filter. The transfer buffer makes its way

through the gel, nitrocellulose paper, and towels by capillary action, taking the DNA with it. The single-

stranded DNA is stopped by the nitrocellulose paper. (4) Blot is incubated with radioactive or fluorescent probes in sealed bag. (5) The positions of the DNA in

the paper directly reflect the positions of the DNA fragments in the gel.

*Blue dress of White House employee Monica Wilson stained with semen of former US President Bill

Clinton!

Southern blots (zoo blots) of various organisms. DNA using a radioactive probe from the Antennapedia gene of Drosophila

melanogaster. Autoradiography shows that Drosophila genes contain several portions that are like Antennapedia genes in structure and that

many organisms contain several genes that will hybridize this radioactive gene fragment, suggesting that Antennapedia-like genes

exist in these organisms. The numbers beside the blots indicate size of bands, in kilobases.

3.MICROARRAYS provide a means to measure & monitor the expression of thousands of genes at

once

...\..\..\genetics\videos\dnaarray.exe

http://highered.mcgraw-hill.com/olc/dl/120078/micro50.swf

DNA microarray analysiscan reveal differences in gene expression in yeast

cells under different experimental conditions. cDNA prepared from mRNA

isolated from wild-type Saccharomyces cells grown

on glucose or ethanol is labeled with different

fluorescent dyes. If a spot is yellow, expression of that gene is the same in cells

grown either on glucose or ethanol. If a spot is green, expression of that gene is greater in cells grown in glucose. If a spot is red,

expression of that gene is greater in cells grown in

ethanol.

3. GENE TARGETING (KNOCK-OUT) EXPERIMENTS- wild type alleles are replaced with mutant ones. There are 2 types of mutations used in these experiments:a. Loss of function mutation- protein product

of the mutant gene is less active than the wild type

b. Gain of function mutation- mutant gene interferes with the function of the wild-type form (mutant can cause receptor activation in the absence of a ligand-receptor complex, or a mutant transcription factor may be active all the time and not respond to regulation)

METHODS OF RECOMBINANT DNA TECHNOLOGY

Knowledge of the molecular biology of cells makes it possible to experimentally move from gene to protein and from protein to

gene!

http://highered.mcgraw-hill.com/olc/dl/120078/bio38.swf

1. Bacterial plasmids are small, circular, self-replicating, extra-chromosomal DNA pieces that can be altered in vitro by inserting or deleting specific sequences, using restriction endonucleases.

Because they can be used to create clones of genes, plasmids are called CLONING VECTORS.

http://highered.mcgraw-hill.com/olc/dl/120078/bio37.swf

...\..\..\genetics\videos\restriction.exe

Transfection- cells are

incubated in solution that makes them

“drink” in cloned DNA

usually mixed with antibiotic

resistance genes

Electropolation-

high-voltage pulse

“pushes” the

cloned DNA into

cell

Microinjection- cloned gene in solution is injected into the cell nucleus. The DNA is injected into the fertilized ovum before the male and female pronuclei have fused, increasing the probability that all of the cells of the organism will harbor the gene.

A “gene gun” is also used which fires plastic bullets filled with DNA-coated metallic pellets. Some may penetrate the nuclei of cells, where the introduced DNA integrates into the DNA of the recipient’s genome.

Transposable element or retroviral vector- cloned DNA is inserted into mobile regions of DNA that has the ability to integrate themselves into the genome of an organism.

Known as jumping genes since they can move about on the chromosome or among chromosomes.

Transgenic mice are produced by random

integration of a foreign gene into the mouse germ line. Foreign DNA

injected into one of the two pronuclei (the male and

female haploidnuclei contributed by the

parents) has a good chance of being randomly integrated into the

chromosomes of thediploid zygote. Because a

transgene is integrated into the recipient genome by

non-homologous recombination, it does not disrupt endogenous genes.

After birth, tissue samples of the young are assessed for the presence of the desired gene. DNA from germ line cells is given special attention.

If the novel gene is present in these cells, the animal and its stem cells can be used as a founder for breeding.

Such animal models allow researchers to test therapeutic compounds and study the molecular basis of given diseases.

Mouse disease models now exist for cystic fibrosis, beta-thalassemia, atherosclerosis, retinoblastoma, and Duchenne muscular dystrophy.

Somatic cells may also be grown in cell culture and genetically modified by fusion with the enucleated egg.

With donor DNA for cloning derived from cultured recombinant cells, it becomes possible to carry out specific genetic modifications and introduce the modified genes into animals and plants.

Isolating embryonic stem (ES) cells and incorporating them into recipient embryos resulting into cells of different

genetic constitution appearing in the same organism (chimera). ES cells treated as transgenics have been useful in determining how genes are regulated during

development of mice.

To analyze the role of BMP7 in development, a bacterial gene for neomycin resistance is inserted into BMP7, destroying its ability to function.

The mutant BMP7 genes are inserted into neomycin sensitive ES cells, heterozygous ES cells are then microinjected into mouse blastocysts, resulting to chimeras which are then mated to wild-type mice.

Heterozygous mice are inbred, producing mutant mice which lacked eyes and kidneys.

Gene targeting makes transgenic mice that are missing specific genes. In this way gene

targeting can be used to analyze the roles of particular genes during mammalian

development.

In the absence of the BMP7 protein, cells that form the eyes & kidneys stop dividing and

die.

...\..\..\genetics\videos\gene targeting.exe

Today, transgenics are

becoming such a common

commodity. GMO technology comes up with new wonders

every now and then, it is not surprising to have one of these in the

future!

4.ANTI- SENSE RNA- interrupts translation of mRNA to protein by introducing single strands of RNA targeted to bind with the mRNA

Generated by cloning DNA into vectors with promoters at both ends of the inserted gene.

When incubated with RNA polymerase and NTPs, the promoter will transcribe the message in the wrong direction.

Transgenic tomatoes have been constructed that carry in their genome an artificial gene that is transcribed into an antisense RNA complementary to the mRNA for an enzyme involved in ethylene production. These tomatoes make only 10% of the normal amount of the enzyme.

This antisense RNA will bind to a normal

cellular message. mRNA produced by this gene will also be degraded.

Results are similar to knock-out experiments where the expression of

a cellular gene is experimentally shut off.

The double-stranded RNA molecules are

recognized by an RNase and degraded into short

fragments.

RNA-dependent RNA polymerase can amplify these fragments, which can be transmitted to

progeny cells.

http://www.dnalc.org/resources/animations/cloning101.html

http://www.dnalc.org/resources/animations/gelelectrophoresis.html

http://www.dnalc.org/resources/animations/pcr.html

http://learn.genetics.utah.edu/content/labs/extraction/

http://learn.genetics.utah.edu/content/labs/microarray/

http://www.dnalc.org/resources/animations/dnaarray.html