DNA Techniques

Dr. Zeyad Akawi Jreisat M.D, M.A, Ph.D

The Potential Use of these Techniques

• Diagnosis:

– Test for DNA sequence variation are more sensitive than many other techniques, such as enzyme assays, Therefore permit recognition of disease at earlier stage

– Identifying carriers of inherited diseases so they can receive appropriate counseling

• Prevention:

– Vaccination (prevention of hepatitis)

• Treatment:

– Diabetes and factor VIII for the treatment of hemophilia

– Gene therapy

• Others:

– Determine family relationships

– Help identifying perpetrators of the crime

Restriction Endonucleases

• Restriction Endonucleases Cleave DNA Molecules at Particular Sites

• Most naturally occurring DNA molecules are much larger than can readily be managed, or analyzed, in the laboratory. If we are to study individual genes and individual sites on DNA, the large DNA molecules found in cells must be broken into manageable fragments. This can be done using restriction endonucleases that cleave DNA at particular sites by recognizing specific sequences.

• Restriction enzymes used in molecular biology typically recognize short (4 to 8 bp) target sequences, usually palindromic, and cut at a defined position within those sequences.

• Of note, some restriction enzymes are sensitive to methylation. That is, methylation of a base (or bases) within a recognition sequence inhibits enzyme activity at that site.

• Restriction enzymes differ not only in the specificity and length of their recognition sequences but also in the nature of the DNA ends they generate. Thus, some enzymes, such as HpaI, generate flush or “blunt” ends; others, such as EcoRI, HindIII, and PstI, generate staggered ends

Restriction Endonucleases

- They are of bacterial origin, They protect bacteria from viruses.

- They permit construction of a new type of genetic map (restriction map: map shows you where each type of RE will cut, its fixed)

- Some hydrolyze the two strands of DNA in a staggered fashion producing sticky or cohesive ends, while others cut both strands symmetrically producing a blunt (Flush) end.

- Each RS has specific site of cutting (ex. G-A) but it works on more than one location on the DNA

- No way to have different enzymes recognizing the same sequence, each RS has its sequence length and specific cutting location

Gel Electrophoresis Separates DNA and RNA Molecules according to Size

• DNA and RNA molecules are separated by the technique called Gel Electrophoresis. Linear DNA molecules separate according to size when subjected to an electric field through jelly-like porous material. Because DNA is negatively charged, when subjected to an electrical field in this way, it migrates through the gel toward the positive pole. The gel matrix acts as a sieve through which DNA molecules pass; large molecules (with a larger effective volume) have more difficulty passing through the pores of the gel and thus migrate through the gel more slowly than do smaller DNAs. This means that once the gels have been electrophoresed or “run” for a given time, molecules of different sizes are separated because they have moved different distances through the gel. After electrophoresis is complete, the DNA molecules can be visualized by staining the gel with fluorescent dyes like ethidium bromide, which binds to DNA and intercalates between the stacked bases. The stained DNA molecules appear as “bands” that each reveal the presence of a population of DNA molecules of a specific size.

• Two alternative kinds of gel matrices are used: polyacrylamide and agarose. Polyacrylamide has high resolving capability but can separate DNAs over only a narrow size range. Thus, electrophoresis through polyacrylamide can resolve DNAs that differ from each other in size by as little as a single base pair but only with molecules of up to several hundred ( just under 1000) base pairs. Agarose has less resolving power than polyacrylamide but can separate DNA molecules of up to tens, and even hundreds, of kilobases.

DNA Gel Electrophoresis

Gel Electrophoresis Apparatus

Sample of gel electrophoresis

Sample of gel electrophoresis

Restriction maps :

http://www.youtube.com/watch?v=8FqMUF96cPE - Permit the routine preparation of defined segments of DNA : which enzyme should we use to cut to

get the wanted gene?

- They used to demonstrate sequence diversity:If we expect an enzyme to cut a specific sequence and it doesn’t cut it, there is a problem, and that sequence differs (polymorphism) Ex. 2 people with X gene, the enzyme cuts the X gene in the first person and doesn’t cut the X gene in the second one, why? X gene differs (polymorphism)

- They can be used to detect deletion mutations.By Taking 2 different DNA and restrict them to the same enzyme, and observe the cut parts, if they differ there is a deletion mutation

- They are crucial for cloning and for sequencing genes and their flanking DNA regions : cut specefic sites we want to clone

Restriction Maps

Restriction mapping of the pBR322 plasmid

Restriction mapping of the pBR322 plasmid

DNA Hybridization Can Be Used to Identify Specific DNA Molecules

• The capacity of denatured DNA to reanneal (i.e., to re-form base pairs between complementary strands) allows for the formation of hybrid molecules when homologous, denatured DNAs from two different sources are mixed with each other under the appropriate conditions of ionic strength and temperature. This process of base pairing between complementary single-stranded polynucleotides is known as hybridization.

• Many techniques rely on the specificity of hybridization between two DNA molecules of complementary sequence. For example, this property is the basis for detecting specific sequences within complicated mixtures of nucleic acids. In this case, one of the molecules is a probe of defined sequence—either a purified fragment or a chemically synthesized DNA molecule. The probe is used to search mixtures of nucleic acids for molecules containing a complementary sequence. The probe DNA must be labeled so that it can be readily located once it has found its target sequence.

Labeling by Radioactive( P32,S35) or chemically (Fluorescent)Radioactive cause less damage than chemical labeling

DNA hybridization

• DNA hybridization can be used to identify specific DNA molecules

• Probes are needed: either a purified fragment or a chemically synthesized DNA molecule. The probe is used to search mixtures of nucleic acids. probes should be labeled.

• Southern blot

• Northern blot

DNA labeling

There are three basic methods for labeling DNA. The first involves adding a label to the end of an intact DNA molecule. Thus, for example, the enzyme polynucleotide kinase adds the gamma-phosphate from ATP to the 5’-OH group of DNA. If that phosphate is radioactive, this process labels the DNA molecule to which it is transferred. Labeling by incorporation (the other mechanism) involves synthesizing new DNA in the presence of a labeled precursor. This approach is often performed by using PCR with a labeled precursor, or even by hybridizing short random hexameric oligonucleotides to DNA and allowing a DNA polymerase to extend them. The labeled precursors are most commonly nucleotides modified with either a fluorescent moiety or radioactive atoms. In addition, DNA can be labeled by so called nick translation.nicking (cutting) DNA with specific enzymes and introducing labeled (florescent) nucleotide.

• DNA labeled with fluorescent precursors can be detected by illuminating the DNA sample with appropriate wavelength UV light and monitoring the longer-wavelength light that is emitted in response. Radioactively labeled precursors typically have radioactive P32 or S35 incorporated into the alpha-phosphate of one of the four nucleotides. This phosphate is retained in the product DNA. Radioactive DNA can be detected by exposing the sample of interest to X-ray film or by photomultipliers that emit light in response to excitation by the beta particles emitted from P32and S35.

Nick translation to label DNA probe

Labeling using primers

Labeling using non-radioactive chemicals

Hybridization Probes Can Identify Electrophoretically Separated DNAs and RNAs

• It is often desirable to monitor the abundance or size of a particular DNA or RNA molecule in a population of many other similar molecules. For example, this can be useful when determining the amount of a specific mRNA that is expressed in two different cell types or the length of a restriction fragment that contains the gene being studied. This type of information can be obtained using blotting methods that localize specific nucleic acids after they have been separated by electrophoresis.

• Suppose that the yeast genome has been cleaved with the restriction enzyme EcoRI and the investigator wants to identify or know the size of the fragment that contains the gene of interest. When stained with ethidium bromide, the thousands of DNA fragments generated by cutting the yeast genome are too numerous to resolve into discretely visible bands, and they look like a smear centered around 4 kb. The technique of Southern blot hybridization (named after its inventor Edward Southern) will identify within the smear the size of the particular fragment containing the gene of interest.

Southern Blot Hybridization http://www.youtube.com/watch?v=RsMsAiNVIGw

• In this procedure, the cut DNA is separated by gel electrophoresis, and the gel is soaked in alkali to denature the double-stranded DNA fragments. These fragments are then transferred from the gel to a positively charged membrane to which they adhere, creating an imprint, or “blot,” of the gel. During the transfer process, the DNA fragments are bound to the membrane in positions that mirror their corresponding positions in the gel after electrophoresis. After DNAs of interest are bound to the membrane, the charged membrane is incubated with a mixture of nonspecific DNA fragments to saturate all of the remaining binding sites on the membrane. Because the DNA in this mixture is randomly distributed on the membrane and, if chosen properly, will not contain the sequence of interest (e.g., from a different organism than the probe DNA), it will not interfere with subsequent detection of a specific gene.

• The DNA bound to the membrane is then incubated with probe DNA containing a sequence complementary to a sequence within the gene of interest. Because all of the nonspecific binding sites on the membrane are occupied with unrelated DNA, the only way that the probe DNA can associate with the membrane is by hybridizing to any complementary DNA present on the membrane. This probing is performed under conditions of salt concentration and temperature close to those at which nucleic acids denature and renature.

Southern Blot Hybridization…cont

• Under these conditions, the probe DNA will hybridize tightly to only its exact complement. Often the probe DNA is in high molar excess compared with its immobilized target on the filter, thereby favoring probe hybridization rather than reannealing of the denatured DNA on the blot. In addition, the immobilization of the denatured DNA on the filter tends to interfere with renaturation.

• A variety of films or other media sensitive to the light or electrons emitted by the labeled DNA can detect where on the blot the probe hybridizes. For example, when a radioactively labeled probe DNA is used and X-ray film is exposed to the filter and then developed, an autoradiogram is produced in which the pattern of exposure on the film corresponds to the position of the hybrids on the blot.If we end up with one band, its called HIGH stingency, more than one, we call it low stringency

Northern Blot Hybridization

• Used to identify a particular mRNA in a population of RNAs. Because mRNAs are relatively short (typically ,5 kb), there is no need for them to be digested with any enzymes (there are only a limited number of specific RNA-cleaving enzymes). Otherwise, the protocol is similar to that described for Southern blotting. The separated mRNAs are transferred to a positively charged membrane and probed with a probe DNA of choice. (In this case, hybrids are formed by base pairing between complementary strands of RNA and DNA).

• An investigator might perform northern blot hybridization to ascertain the amount of a particular mRNA present in a sample rather than its size. This measure is a reflection of the level of expression of the gene that encodes that mRNA. Thus, for example, one might use northern blot hybridization to ask how much more mRNA of a specific type is present in a cell treated with an inducer of the gene in question compared with an uninduced cell. As another example, northern blot hybridization might be performed to compare the relative levels of a particular mRNA (and hence the expression level of the gene in question) among different tissues of an organism. Because an excess of DNA probe is used in these assays, the amount of hybridization is related to the amount of mRNA present in the original samples, allowing the relative amounts of mRNA to be determined.

Southern blot

Southern blotting

Isolation of Specific Segments of DNA

• Much of the molecular analysis of genes and their function requires the separation of specific segments of DNA from much larger DNA molecules and their selective amplification. Isolating a large amount of a single pure DNA molecule facilitates the analysis of the information encoded in that particular DNA molecule. Thus, the DNA can be sequenced and analyzed, or it can be cloned and expressed to allow the study of its protein product.

• Recombinant DNA molecules can be created and used to alter the expression of a particular gene (e.g., by fusing its coding sequence to a heterologous promoter). Alternatively, purified DNA sequences can be recombined to generate DNAs that encode so-called fusion protein that is, hybrid proteins made up of parts derived from different proteins. The techniques of DNA cloning and amplification by PCR have become essential tools in asking questions regarding the control of gene expression, maintenance of the genome, and protein function.

Note : this part will be explained in slide number 47

DNA cloning

• The ability to construct recombinant DNA(2 DNA molecules from different origins) molecules and maintain them in cells is called DNA cloning. This process typically involves a vector that provides the information necessary to propagate the cloned DNA in the replicating host cell. Key to creating recombinant DNA molecules are the restriction enzymes that cut DNA at specific sequences and other enzymes that join the cut DNAs to one another. By creating recombinant DNA molecules that can be propagated in a host organism, a particular DNA fragment can be both purified from other DNAs and amplified to produce large quantities.

• Cloning DNA in plasmid vector: Once DNA is cleaved into fragments, it typically needs to be inserted into a vector for propagation. That is, the DNA fragment must be inserted into a second DNA molecule (the vector) to be replicated in a host organism. The most common host used to propagate DNA is the bacterium E. coli. Many common vectors are small (3 kb) circular DNA molecules called plasmids. These molecules were originally derived from extrachromosomal circular DNA molecules that are found naturally in many bacteria and single-cell eukaryotes.

http://www.youtube.com/watch?v=MIfDx417SDs

DNA cloning…cont

• A fragment of DNA, generated by cleavage with EcoRI, is inserted into the plasmid vector linearized by the same enzyme. Once ligated, the recombinant plasmid is introduced into bacteria by transformation. Cells containing the plasmid can be selected by growth on the agar plates that contain growth media including antibiotic to which the plasmid confers resistance.

• Some vectors not only allow the isolation and purification of a particular DNA but also drive the expression of genes within the insert DNA. These plasmids are called expression vectors and have transcriptional promoters, derived from the host cell, immediately adjacent to the site of insertion. If the coding region of a gene (without its promoter) is placed at the site of insertion in the proper orientation, then the inserted gene will be transcribed into mRNA and translated into protein by the host cell. Expression vectors are frequently used to express heterologous or mutant genes to assess their function. They can also be used to produce large amounts of a protein for purification. In addition, the promoter in the expression vector can be chosen such that expression of the insert is regulated by the addition of a simple compound to the growth media (e.g., a sugar or an amino acid)

DNA Cloning

In standard cloning protocols, the cloning of any DNA fragment essentially involves seven steps:

1- Choice of host organism and cloning vector,2- Preparation of vector DNA,3- Preparation of DNA to be cloned, 4- Creation of recombinant DNA, 5- Introduction of recombinant DNA into the host

organism,6- Selection of organisms containing recombinant

DNA, 7- Screening for clones with desired DNA inserts and

biological properties.

Applications of recombinant DNA technology

- Biotechnology- Medicine- Research

Applications of recombinant DNA technology

Medicine:- Recombinant human insulin: before they used to isolate the insulin from

pancreatic tissue, now they use DNA recombination technology

- Recombinant human growth hormone (HGH, somatotropin)- Recombinant blood clotting factor VIII- Recombinant hepatitis B vaccine- Diagnosis of infection with HIV: each of the three widely used

methods for diagnosing HIV infection has been developed using recombinant DNA. The antibody test (ELISA or western blot) uses a recombinant HIV protein to test for the presence of antibodies that the body has produced in response to an HIV infection. The DNA test looks for the presence of HIV genetic material using reverse transcriptase polymerase chain reaction (RT-PCR). Development of the RT-PCR test was made possible by the molecular cloning and sequence analysis of HIV genomes.

Recombinant DNA Technology

- It is a combination of recombinant DNA, replication, separation and identification that permits the production of large quantities of purified DNA fragments.

- Cloning vectors: Plasmids, Bacteriophage-, Cosmid, and yeast cloning vectors.

we choose the vector depending on the SIZE of DNA fragment you want to cloneYeast > Cosmid > Bacteriophage- > plasmid

- Plasmids: have the ability to confer antibiotic resistance to the bacterium, used to select the bacteria that has the recombinant DNAEx. Bacteria selection : adding antibiotic solution to all Bacteria .Bacteria with recombinant DNA with plasmid vector tend to have antibiotic –resistant feature used to help it in surviving the antibiotic solution thus it will be selected

- Most vectors contain an inserted sequence of DNA termed polylinker, restriction site bank or polycloning site. The site where the RE make the specific cuts

Desirable features of a plasmid vector

- Relatively low molecular weight (3-5kb) to accommodate larger fragments of DNA of interest

- Several different restriction endonuclease sites.

- Multiple selectable markers (antibiotic-resistance) , to aid in selecting bacteria with recombinant DNA molecule.

- High rate of replication.

pBR322 Plasmid

Formation of Recombinant DNA

The action of a restriction enzyme and the production of recombinant DNA

Construction of a bacterial expression vector

For amplification purposes, you can use or better to use only one type of restriction enzyme. Why ? – because we don’t care how we introduce the DNA into the plasmid vector (either 3'-5' or 5'-3') ; the way the DNA introduce in the vector will not affect the process of replication because we have origin of replication and when the plasmid starts to replicate (because its highly replicatable ) it well replicate all DNA that contained in that plasmid including the DNA of interest.

When we need to express the gene of interest to get protein, this way we cannot introduce the piece of cDNA anywhere in the plasmid ,,we have to have an orientation for this insertion. Why orientation ? because we cannot express a given protein unless we have the gene of interest located downstream the promoter of a given DNA that included in the plasmid or the plasmid promoter the directional cloning, we have the plasmid which should have a promoter

in this situation we have for the plasmid two restriction sites (site A , site B ) this means thatyou have to use two different restriction enzymes in order to make sure that the piece of DNA that inserted is inserted in a given orientation not in the other.

In this situation we use the Lac Z with its promoter ,, what is the Lac Z ? its Lac operon and it presents in E.coli (remember Lac Z ,, Lac A,, Lac Y) Lac Z with its promoter will be introduced in this plasmid why? Because the bacteria recognize the Lac Z promoter and it is very active promoter in bacteria and this will get benefit out of this in term of amplification more and more, production of the gene of interest that located downstream of the promoter and the Lac Z gene,, By this you will form the protein from the Lac Z gene and you form the protein for the DNA of interest ,,these together we call fusion protein – see slide 35, now you will understand it insha allah

Example of the usage of recombinant DNA technology in medicine

Insulin-amplification

First you get a DNA from tiny pancreatic tissue

Bacteria only can produce proteins out of DNA without introns, so

if you want to amplify the insulin gene without concern about expression you can introduce the whole gene and amplify it ..

for expression purposes you have to be exact in direction and have cDNA(DNA without introns) instead of whole DNA ,,we will talk about how we make cDNA

from RNA 'from DNA that will result in mature RNA' we have the human insulin

complementary DNA (cDNA) is DNA synthesized from a messenger RNA (mRNA) template in a reaction catalysed by the enzymes reverse transcriptase and DNA polymerase

DNA sequencing by SangerWatch this amazing video for this method:

http://www.youtube.com/watch?v=bEFLBf5WEtc

Up largestDown smallest

DNA Sequencing Gel

DNA Sequencing comparing point normal with mutation

DNA sequencingThe outcome sequence you get is the sequence of the polymerized one, if you want the sequence of the DNA of interest you take the complementary one

Restriction Fragment Length Polymorphismhttp://www.youtube.com/watch?v=d2jEyO3hqR0

Restriction Fragment Length Polymorphism

Restriction Fragment Length Polymorphism

Southern blot hybridization of a specific RFLP

Southern blot for PKU