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Gene Analysing:

DNA biochemistry:
There are many steps in the process of genetic manipulation of DNA from cutting it to amplifying the recombinant molecule which has been produced.
In order to isolate biochemically useful DNA the sequence should be as long as possible. The DNA must first be obtained from the required cells. If eukaryotic cells are being used this is a relatively simple process as they cells are surrounded by only a lipid membrane. Lipid is soluble in detergent and so adding detergent to eukaryotic cells will lyse them. A commonly used detergent is called sodium dodecyl sulphate (SDS). Enzymes such as nucleases, which lead to the degradation of genetic material also first, have to be inactivated and SDS does this too.
When using prokaryotic cells the polysaccharide coating has to be first removed using the enzyme lysozyme, then the SDS can be used to lyse the cell etc. Insoluble material is removed by centrifuge leaving the required genetic material, DNA (and RNA) along with protiens. When DNA has be removed, the genetic material has to be purified, for genetic material of high molecular weight, Phenol can be added to the insoluble solution that has been obtained. This is mixed which creates an emulsion of all the tube contents. Centrifugation of this will lead to the formation of two layers, with the genetic material in the upper phase, the phenol in the lower and the insoluble protein on the interface between the two layers. By removing the uppermost layer the genetic material can be successfully removed. This upper layer which has been removed still contains RNA, which can be separated from the DNA by "buoyant density centrifugation". In this process the genetic mixture is placed in to a centrifuge tube with Caesium chloride at high concentration. Once a gradient is set up the concentration in the centre of the tube will enable the DNA to concentrate there by equilibrium. This tube is centrifuged for up to forty hours at 40,000 rpm. At such a high speed the centrifugal forces imparted in to the solution, will cause the heavy caesium ions to move towards the outside of the tube. This causes density and concentration gradients to be set up, giving rise to the DNA (which has a slightly lower density than RNA), to concentrate towards the middle (due to the differing sedimentation coefficients of the molecules). The denser RNA moves towards the sides of the tube (or the bottom depending on tube orientation).
The DNA obtained can be processed in slightly varying ways. If a high molecular weight is not required then adding a large concentration of ethanol, which is then centrifuged to form a pellet, can precipitate the DNA. The remaining ethanol is then removed by drying and the remaining pellet is re-dissolved in the required buffer. For situations where large DNA fragments are required, the DNA is not precipitated using alcohol, as the high pressures imparted by the centrifugation can lead to the molecules being sheared.
'Restriction endonucleases' or 'Restriction enzymes' are the backbone behind recombinant DNA technology. The power of these enzymes lies in their ability to cut DNA in to defined fragments at specific sequences of the genetic coding. Restriction enzymes originate from many different forms of bacteria. It had been noted that some forms of bacteriophage grew in forms of E.coli but not in apparently similar forms. It was found that these bacteria were producing an enzyme, which had the ability to digest DNA at specific sites on DNA, another complimentary enzyme was found that had the ability of methylating the bacterial DNA at the same site. This methylation protected the bacterial genome from being digested by an endonuclease it produced, but the endonuclease still degraded any unmethylated DNA in the bacterial cell such as that of invading viral DNA, this is known as 'host restriction'. Many restriction enzymes have been found, in many different bacteria, all of which have the ability of protecting bacteria against viral DNA being integrated in to its own genetic sequence. Each of these enzymes recognised and 'restricted' a different and specific genetic sequence.
This wide range of different enzymes (and therefore different cutting sites) enables us to manipulate DNA in such a simple yet specific way. Apart from each enzyme cutting at a different specific site, another property of these endonucleases is their differing properties in producing cutting sites. Whilst some leave 'blunt' ends with no DNA overhang others produce cohesive or 'sticky' ends which aid in the re-ligation of genetic material. Although some enzymes such as BamH and BglII recognise different cutting sites, the cohesive ends they produce are both identical, which may be a useful property if engineering with more than one enzyme or genetic insert. The names of these endonucleases are determined from the bacteria from which they are obtained; for example EcoR1 is derived from E.coli. The figure 1 in EcoR1 indicates that this was the first enzyme to be isolated from E.coli.
The most important qualities of restriction enzymes are their digestive properties. Most commonly used enzymes recognise a short sequence of bases (usually 4-6 bases long). The number of bases in a recognised sequence is important as if an enzyme recognised just one base i.e. adenine, then on average the DNA would be able to be cut at every fourth base. For any sequence of six bases to randomly occur the enzyme would statistically only find a cutting site every four thousand and ninety six bases. Although the sequence of bases in genetic material are not randomly distributed as genes are related and sequences conserved this specific site recognition lowers the number of cutting sites on a genome considerably. On a bacterium with a small chromosome, there may only be one or two such cutting sites. When an enzyme such as EcoR1 cuts the DNA, a phosphate group stays on the 5' end of the DNA and an OH group resides on the 3' end. If the cut in the DNA does not create blunt ends, the ends of the DNA are regarded as sticky because of their ability to briefly pair again, however at normal room temperature this pairing is unstable and short lived.
Once DNA has been restricted, using an enzyme or number of enzymes, each fragment that has been created may be of an unknown length and the total number of fragments may be unknown. To resolve these problems a technique named 'agarose gel electrophoresis' is employed. The agarose gel used in this technique has the property of becoming a liquid when heated and setting in to a jelly like substance when it cools because of the cross-linking of large polysaccharide molecules in it. By setting up a difference in voltage across the gel, it is possible to impel fragments of negatively charged DNA molecules to move through the agarose, away from the cathode and towards the positively charged anode. Because of the cross-linking in the gel, the larger molecules have a greater resistance towards moving through the gel than the small ones so they progress more slowly. The distance the DNA has moved is measured from the well it was loaded in to (at the cathode end), to the centre of the band at the end of the run.
Because different DNA and different enzymes can yield fragments of drastically varying size, it is important to separate out the bands as much as possible without losing any or letting any merge.
To do this the run can take place using currents of different strengths and by using agarose which has different amounts of cross-linking so giving different specific resistance's to the material travelling through it. This process helps to clarify each band and to obtain accurate, measurable results. When the DNA is pipetted in to the wells, it goes in with a running dye, which will also run towards the anode. Due to its size it will move faster than the DNA so when it reaches the end the run can
be terminated and no bands should have been lost. Once the run is complete, it is important to visualise the positions of the bands of the genetic material. The gel is removed and placed in a bath of ethidium bromide; this substance has both the property of fluorescing under ultra violet light and of getting between the bases on a DNA molecule. The DNA in the gel will incorporate the ethidium bromide and so fluorescent bands on the gel will determine their positions when exposed to UV light. These band positions are recorded and used to determine the size of each fragment. As ethidium bromide gets between the bases of DNA it is a potential mutagen and therefore a potential carcinogen and exposure to it should be kept as low as possible. The mobilities of DNA fragments of a determined size are plotted against the log10 of their size. By this method the size of unknown fragments can be determined by reading the distance they have moved against the equivalent log size on the graph.
As well as cutting DNA using endonucleases, an important aspect of genetic manipulation is the ability to join fragments of genetic material together. The cohesive ends that remain after the digestion with restriction enzymes are naturally complimentary, but are inherently unstable under normal conditions and will not rejoin easily. By using the enzyme 'DNA ligase', the cohesive ends left by the digestion with the enzyme will be able to join again creating one large fragment rather than many smaller ones. The ligase enables the 3' OH group to covalently bond to the 5' phosphate group re-ligating the molecule together. This will only work if the vector DNA and the DNA have both been digested using the same enzyme, or enzymes that produce the same sticky ends. Ligase is often obtained from E.coli, which has been infected with the T4 bacteriophage, this requires ATP as a co-factor but the T4 ligase produced has the ability to join blunt ends well.
Now that the recombinant DNA ligated in to a suitable vector such as a plasmid or bacteriophage it is important to introduce it in to a system that will amplify it, such as a bacterium. Under normal conditions bacteria will not take up these free plasmids and have to become 'competent' to do so, this process is known as 'transformation'. Treating the bacteria with a solution of calcium chloride and then 'heat shocking' does this. The plasmid and bacterial solutions are mixed and some of the bacteria will take up the plasmids. As less than one percent usually do so; each bacterium should take up a maximum of one plasmid and should only contain one recombinant molecule. By using a plasmid containing a gene that codes for resistance to an antibiotic such as the Amp gene (which gives resistance to ampicillin), it can be determined which bacteria have taken up a plasmid. The bacteria are plated out at low concentration on an agar jelly containing the said antibiotic, and if none of them had resistance before the treatment only the ones containing the plasmid and resistance gene will survive. As each bacterium forms a colony, each cell in it will contain a single type of DNA molecule; this process is known as DNA cloning as each cell in a colony is identical to the rest.
Increasingly there are many uses for such genetic manipulation, by inserting the gene that codes for human insulin in to bacteria, they will begin to create the insulin protein and provide a useful drug for those with diabetes. Another approach to genetic engineering is the manipulation of human somal cells, such as in 'gene therapy'. Sufferers of cystic fibrosis have inherited two copies of a defective gene that should control the mucous glands mucous production leading to a build up phlegm on the lungs. An experimental approach to dealing with such a situation uses a cold virus, which has been genetically manipulated to deliver a working version of the gene to the sufferer's lungs. This gene should then begin to code for the protein that is normally not produced by patients with cystic fibrosis.
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restrict   [Show phonetics]
verb [T]
to limit the movements or actions of someone, or to limit something and reduce its size or prevent it from increasing

restriction

1. The process with which foreign DNA that has been introduced into a prokaryotic cell becomes ineffective.

 

Southerns, Northerns, Westerns, & Cloning:
"Molecular Searching" Techniques

http://web.mit.edu/esgbio/www/rdna/rdna.html

Written by Brian White, MIT. Copyright 1995.

These are techniques for analyzing cellular macromolecules: DNA, RNA, and protein. These sections will describe how they work and how they can be used as analytical tools.

For Further Reading:
Gel electrophoresis and Southern blotting are described in Purves, Oriens, and Heller pp. 315-8. DNA-RNA hybridization is described on pp. 288-9.

Theory: Complementarity and Hybridization

Molecular searches use one of several forms of complementarity to identify the macromolecules of interest among a large number of other molecules. Complementarity is the sequence-specific or shape-specific molecular recognition that occurs when two molecules bind together. For example: the two strands of a DNA double-helix bind because they have complimentary sequences; also, an antibody binds to a region of a protein molecule because they have complimentary shapes.

Complementarity between a probe molecule and a target molecule can result in the formation of a probe-target complex. This complex can then be located if the probe molecules are tagged with radioactivity or an enzyme. The location of this complex can then be used to get information about the target molecule.

In solution, hybrid molecular complexes (usually called hybrids) of the following types can exist (other combinations are possible):

* 1) DNA-DNA. A single-stranded DNA (ssDNA) probe molecule can form a double-stranded, base-paired hybrid with a ssDNA target if the probe sequence is the reverse complement of the target sequence.

* 2) DNA-RNA. A single-stranded DNA (ssDNA) probe molecule can form a double-stranded, base-paired hybrid with an RNA (RNA is usually a single-strand) target if the probe sequence is the reverse complement of the target sequence.

* 3) Protein-Protein. An antibody probe molecule (antibodies are proteins) can form a complex with a target protein molecule if the antibody's antigen-binding site can bind to an epitope (small antigenic region) on the target protein. In this case, the hybrid is called an 'antigen-antibody complex' or 'complex' for short.

There are two important features of hybridization:

1) Hybridization reactions are specific - the probes will only bind to targets with complimentary sequence (or, in the case of antibodies, sites with the correct 3-d shape).

2) Hybridization reactions will occur in the presence of large quantities of molecules similar but not identical to the target. That is, a probe can find one molecule of target in a mixture of zillions of related but non-complementary molecules.

These properties allow you to use hybridization to perform a molecular search for one DNA molecule, or one RNA molecule, or one protein molecule in a complex mixture containing many similar molecules.

These techniques are necessary because a cell contains tens of thousands of genes, thousands of different mRNA species, and thousands of different proteins. When the cell is broken open to extract DNA, RNA, or protein, the result is a complex mixture of all the cell's DNA, RNA, or protein. It is impossible to study a specific gene, RNA, or protein in such a mixture with techniques that cannot discriminate on the basis of sequence or shape. Hybridization techniques allow you to pick out the molecule of interest from the complex mixture of cellular components and study it on its own.

Basic Definitions

Blots are named for the target molecule.

Southern Blot
DNA cut with restriction enzymes - probed with radioactive DNA.

Northern Blot
RNA - probed with radioactive DNA or RNA.

Western Blot
Protein - probed with radioactive or enzymatically-tagged antibodies.

Overview

The formation of hybrids in solution is of little experimental value - if you mix a solution of DNA with a solution of radioactive probe, you end up with just a radioactive solution. You cannot tell the hybrids from the non-hybridized molecules. For this reason, you must first physically separate the mixture of molecules to be probed on the basis of some convenient parameter.

These molecules must then be immobilized on a solid support, so that they will remain in position during probing and washing. The probe is then added, the non-specifically bound probe is removed, and the probe is detected. The place where the probe is detected corresponds to the location of the immobilized target molecule. This process is diagrammed below:

In the case of Southern, Northern, and Western blots, the initial separation of molecules is done on the basis of molecular weight. (Cloning uses a different technique.)

In general, the process has the following steps, detailed below:

Gel Electrophoresis

This is a technique that separates molecules on the basis of their size.

First, a slab of gel material is cast. Gels are usually cast from agarose or poly-acrylamide. These gels are solid and consist of a matrix of long thin molecules forming sub-microscopic pores. The size of the pores can be controlled by varying the chemical composition of the gel. The gel is cast soaked with buffer.

The gel is then set up for electrophoresis in a tank holding buffer and having electrodes to apply an electric field:



The pH and other buffer conditions are arranged so that the molecules being separated carry a net (-) charge so that they will me moved by the electric field from left to right. As they move through the gel, the larger molecules will be held up as they try to pass through the pores of the gel, while the smaller molecules will be impeded less and move faster. This results in a separation by size, with the larger molecules nearer the well and the smaller molecules farther away.

Note that this separates on the basis of size, not necessarily molecular weight. For example, two 1000 nucleotide RNA molecules, one of which is fully extended as a long chain (A); the other of which can base-pair with itself to form a hairpin structure (B):



As they migrate through the gel, both molecules behave as though they were solid spheres whose diameter is the same as the length of the rod-like molecule. Both have the same molecular weight, but because B has secondary (2') structure that makes it smaller than A, B will migrate faster than A in a gel. To prevent differences in shape (2' structure) from confusing measurements of molecular weight, the molecules to be separated must be in a long extend rod conformation - no 2' structure. In order to remove any such secondary or tertiary structure, different techniques are employed for preparing DNA, RNA and protein samples for electrophoresis.

Preparing DNA for Southern Blots
DNA is first cut with restriction enzymes and the resulting double-stranded DNA fragments have an extended rod conformation without pre-treatment.
Preparing RNA for Northern Blots
Although RNA is single-stranded, RNA molecules often have small regions that can form base-paired secondary structures. To prevent this, the RNA is pre-treated with formaldehyde.
Preparing Proteins for Western Blots
Proteins have extensive 2' and 3' structures and are not always negatively charged. Proteins are treated with the detergent SDS (sodium dodecyl sulfate) which removes 2' and 3' structure and coats the protein with negative charges.
If these conditions are satisfied, the molecules will be separated by molecular weight, with the high molecular weight molecules near the wells and the low molecular weight molecules far from the wells. The distance migrated is roughly proportional to the log of the inverse of the molecular weight (the log of 1/MW). Gels are normally depicted as running vertically, with the wells at the top and the direction of migration downwards. This leaves the large molecules at the top and the smaller molecules at the bottom. Molecular weights are measured with different units for DNA, RNA, and protein:
  • DNA: Molecular weight is measured in base-pairs, or bp, and commonly in kilobase-pairs (1000bp), or kbp.
  • RNA: Molecular weight is measured in nucleotides, or nt, and commonly in kilonucleotides (1000nt), or knt. [Sometimes, bases, or b and kb are used.]
  • Protein: Molecular weight is measured in Daltons (grams per mole), or Da, and commonly in kiloDaltons (1000Da), or kDa.

On most gels, one well is loaded with a mixture of DNA, RNA, or protein molecules of known molecular weight. These 'molecular weight standards' are used to calibrate the gel run and the molecular weight of any sample molecule can be determined by interpolating between the standards. Below is a gel stained with a dye: a colored molecule which binds to a specific class of macromolecules in a sequence-independent manner (probes bind in a sequence-dependent manner).

Sample 1 contains only one size class of macromolecule - it could be a plasmid, a pure mRNA transcript, or a purified protein. In this case, you would not have to use a probe to detect the molecule of interest since there is only one type of molecule present. Blotting is usually necessary for samples that are not complex mixtures. By interpolation, its molecular weight is roughly 3.

Sample 2 is what a sample of total DNA cut with a restriction enzyme, total cellular RNA, or total cellular protein would look like in a gel stained with a sequence-independent stain. There are so many bands that it is impossible to find the one we are interested in. Without a probe (which acts like a sequence-dependent stain) we cannot get very much information from a sample like this.



Different stains and staining procedures are used for different classes of macromolecules:

Staining DNA
DNA is stained with ethidium bromide (EtBr), which binds to nucleic aids. The DNA-EtBr complex fluoresces under UV light.

Staining RNA
RNA is stained with ethidium bromide (EtBr), which binds to nucleic aids. The RNA-EtBr complex fluoresces under UV light.

Staining Protein
Protein is stained with Coomassie Blue (CB). The protein-CB complex is deep blue and can be seen with visible light.

Transfer to Solid Support

After the DNA, RNA, or protein has been separated by molecular weight, it must be transferred to a solid support before hybridization. (Hybridization does not work well in a gel.) This transfer process is called blotting and is why these hybridization techniques are called blots. Usually, the solid support is a sheet of nitrocellulose paper (sometimes called a filter because the sheets of nitrocellulose were originally used as filter paper), although other materials are sometimes used. DNA, RNA, and protein stick well to nitrocellulose in a sequence-independent manner.

The DNA, RNA, or protein can be transferred to nitrocellulose in one of two ways:

1) Electrophoresis, which takes advantage of the molecules' negative charge:

2) Capillary blotting, where the molecules are transferred in a flow of buffer from wet filter paper to dry filter paper:

Note: In a Southern Blot, the DNA molecules in the gel are double-stranded, so they must be made single stranded in order for the probe to hybridize to them. To do this, the DNA is transferred using a strongly alkaline buffer, which causes the DNA strands to separate - this process is called denaturation - and bind to the filter as single-stranded molecules. RNA an protein are run in the gels in a state that allows the probe to bind without this pre-treatment.

Blocking

At this point, the surface of the filter has the separated molecules on it, as well as many spaces between the lanes, etc., where no molecules have yet bound. If we added the probe directly to the filter now, the probe would stick to these blank parts of the filter, like the molecules transferred from the gel did. This would result in a filter completely covered with probe which would make it impossible to locate the probe-target hybrids. For this reason, the filters are soaked in a blocking solution which contains a high concentration of DNA, RNA, or protein. This coats the filter and prevents the probe from sticking to the filter itself. During hybridization, we want the probe to bind only to the target molecule.

Preparing the Probe

Radioactive DNA probes for Southerns and Northerns

The objective is to create a radioactive copy of a double-stranded DNA fragment. The process usually begins with a restriction fragment of a plasmid containing the gene of interest. The plasmid is digested with particular restriction enzymes and the digest is run on an agarose gel. Since a plasmid is usually less than 20 kbp long, this results in 2 to 10 DNA fragments of different lengths. If the restriction map of the plasmid is known, the desired band can be identified on the gel. The band is then cut out of the gel and the DNA is extracted from it. Because the bands are well separated by the gel, the isolated DNA is a pure population of identical double-stranded DNA fragments.

The DNA restriction fragment (template) is then labeled by Random Hexamer Labeling.:

1) The template DNA is denatured - the strands are separated - by boiling.

2) A mixture of DNA hexamers (6 nucleotides of ssDNA) containing all possible sequences is added to the denatured template and allowed to base-pair. They pair at many sites along each strand of DNA.

3) DNA polymerase is added along with dATP, dGTP, dTTP, and radioactive dCTP. Usually, the phosphate bonded to the sugar (the a-phosphate, the one that is incorporated into the DNA strand) is synthesized from phosphorus-32 (32P), which is radioactive.

4) The mixture is boiled to separate the strands and is ready for hybridization.

This process is diagrammed below (labeled DNA shown in gray):



This produces a radioactive single-stranded DNA copy of both strands of the template for use as a probe.

Radioactive Antibodies for Westerns

Antibodies are raised by injecting a purified protein into an animal, usually a rabbit or a mouse. This produces an immune response to that protein. Antibodies isolated from the serum (blood) of that rabbit will bind to the protein used for immunization. These antibodies are protein molecules and are not themselves radioactive.

They are labeled by chemically modifying the side chains of tyrosines in the antibody with iodine-125 (125I), which is radioactive. A set of enzymes catalyzes the following reaction:

antibody-tyrosine + 125I- + H2O2 ---------> H2O + 125iodo-tyrosine-antibody

Enzyme-conjugated Antibodies for Westerns

Antibodies against a particular protein are raised as above and labeled by chemically cross-linking the antibody molecules to molecules of an enzyme. The resulting antibody-enzyme conjugate is still able to bind to the target protein.

Hybridization

In all three blots, the labeled probe is added to the blocked filter in buffer and incubated for several hours to allow the probe molecules to find their targets.

Washing

After hybrids have formed between the probe and target, it is necessary to remove any probe that is on the filter that is not stuck to the target molecules. Because the nitrocellulose is absorbent, some of the probe soaks into the filter and must be removed. If it is not removed, the whole filter will be radioactive and the specific hybrids will be undetectable.

To do this, the filter is rinsed repeatedly in several changes of buffer to wash off any un-hybridized probe.

Note: In Southerns and Northerns, hybrids can form between molecules with similar but not necessarily identical sequences (For example, the same gene from two different species.). This property can be used to study genes from different organisms or genes that are mutated. The washing conditions can be varied so that hybrids with differing mismatch frequencies are maintained. This is called 'controlling the 'stringency' - the higher the wash temperature, the more stringent the wash, the fewer mismatches per hybrid are allowed.

Detecting the Probe-Target Hybrids

At this point, you have a sheet of nitrocellulose with spots of probe bound wherever the probe molecules could form hybrids with their targets. The filter now looks like a blank sheet of paper - you must now detect where the probe has bound.

Autoradiography

If the probe is radioactive, the radioactive particles that it emits can expose X-ray film. If you press the filter up against X-ray film and leave it in the dark for a few minutes to a few weeks, the film will be exposed wherever the probe bound to the filter. After development, there will be dark spots on the film wherever the probe bound.

Enzymatic Development

If an antibody-enzyme conjugate was used as a probe, this can be detected by soaking the filter in a solution of a substrate for the enzyme. Usually, the substrate produces an insoluble colored product (a chromogenic substrate) when acted upon by the enzyme. This produces a deposit of colored product wherever the probe bound.

Summary:

The procedure for these three blots is summarized below:





The important properties of the three are shown below:




Cloning a Gene by Hybridization:

In this case, 'to clone the actin gene from humans' means "to end up with a plasmid which contains a fragment of human DNA which includes the actin gene". The usual starting point is a plasmid clone of the actin gene from another organism and human chromosomal DNA. DNA-DNA hybridization is usually used for this.

Although Southern blotting involves DNA-DNA hybridization, it is not a useful procedure for cloning a gene. If we were to cut human DNA with a restriction enzyme and run it on a Southern blot probed with a clone of the actin gene from an other organism, we could construct a restriction map of the human actin gene. However, we can not isolate the human actin gene DNA from either the gel or the filter because at each molecular weight on the gel, there are many bands of the same length but different sequences.

For this reason, separating the DNA fragments by molecular weight is unsuitable. Instead, we separate them by sequence - by making a library, we end up with a collection of plasmids, physically separated, each containing a different fragment of human DNA.

Here is a typical procedure: cloning the human gene for actin, given a clone of the yeast actin gene.

1) Isolate genomic (chromosomal) DNA from human cells.

2) Create a plasmid library of human DNA restriction fragments. This results in a collection of bacterial colonies, each containing a different plasmid with a different inserted piece of human DNA.

3) Plate the colonies on agar plates and let them grow. These are called the master plates.

4) Press a piece of nitrocellulose onto each master plate and lift off. This leaves some of each colony on the plate and a replica on the filter.

5) Break open (lyse) the bacteria on the filter under conditions that make their plasmid DNA single-stranded, and bind the DNA onto the filter. There are now spots of single-stranded plasmid DNA on the filter. These spots correspond to the locations of the colonies of bacteria on the master plate.

6-9) Follow steps 5 through 8 of the Southern Blot, using a labeled restriction fragment of the yeast actin gene from the plasmid as a probe. The result will be a piece of X-ray film with a dark spot corresponding to a place on the filter where DNA from the human actin gene was present. This spot on the filter corresponds to a colony from the master plate.

10) Pick up some of the bacteria from the appropriate colony, grow them in broth and extract their plasmid DNA. It contains a fragment of human DNA containing the actin gene.

It is also possible to clone gene X using an antibody against the protein produced by gene X. In this case, you make an expression library - a library where the vector contains a strong E. coli promoter and an ATG codon. Cells containing these plasmids will produce large amounts of protein from whichever human gene they carry. If you prepare the filter replicas of the library as above, you can probe them with an antibody to find the colony that contains a plasmid that expresses protein X. This plasmid will contain gene X.

Now you're ready to learn how to solve problems!