Molecular Genetic Technology

By Dr. Dalia Shaalan Lecturer of Biochemistry and Molecular Biology

Molecular Genetic Technology

DNA

amplification techniques: In vivo = Cloning using Recombinant DNA technology (genetic engineering) In vitro = PCR

Recombinant DNA technology (genetic engineering)

Recombinant DNA technology

Recombinant DNA technology makes it possible: to isolate genes, to determine their base sequences, to manipulate base sequence and to transfer genes from one species to another.

Recombinant DNA technology

Practical applications of recombinant DNA technology:

I-Production of essential materials for biomedical application: It can supply large amounts of material that could not be obtained by the conventional purification methods (e.g., interferon, tissue plasminogen activating factor). Also, it can provide human material like insulin, growth hormone and vaccines.

II- Molecular analysis of Genetic diseases. III- Gene therapy.

Recombinant DNA technology

Recombinant DNA technology

Requirements: This is a splicing technique that involves: 1- Vectors ( a molecule that can carry foreign DNA). 2- DNA from any species e.g. human, rat...etc. 3- Restriction endonucleases: cutting enzymes. 4- Ligases: joining enzyme.

Recombinant DNA technology

Vectors
A vector is a molecule of DNA to which the fragment of DNA to be cloned is attached. The vector then continues to replicate in the host cell. Types of vectors: Plasmids. Phages (viruses). Cosmids.

A) B) C)

A) Plasmids:

Recombinant DNA technology

Definition: Bacterial plasmids are small, circular, extra segments of duplex DNA. Site: Plasmids are present as single or multiple copies within the cytoplasm of bacterial cells. These DNA segments exist and divide independent of organism's nuclear genes. Function: The genes of plasmids code for certain substances which cause antibiotic resistance of the bacterial cell. Plasmids (because of very small size) can be isolated from one cell and transferred to another cell. Plasmids accept DNA pieces about 6-10 kb long.

B) Phages (viruses): They usually have linear DNA molecules into which foreign DNA can be inserted at several restriction enzyme sites. The chimeric DNA is collected after the phage proceeds through its lytic cycle and produces mature, infective phage particles. A major advantage of phage vectors is that it can accept DNA fragments 10-20 kb long.

Recombinant DNA technology

C) Cosmids: They are plasmids that contain the DNA sequences required for packaging lambda DNA into the phage particle. They can carry chimeric DNA segments about 35-50 kb long.

Recombinant DNA technology

Definition: Restriction endonucleases (RE) are enzymes that cut DNA at precise base sequences producing defined DNA fragments. Over 100 RE are isolated and named according to the bacterial species from which they were isolated e.g. EcoR1. The first letter indicates the genus name, the other two indicate the species name and a roman numeral is used to identify the enzyme. EcoR1 was the first enzyme isolated from Escherichia coli (E.coli).

Restriction endonucleases

Recombinant DNA technology

They were discovered in the bacterial cells which use RE to destroy invading foreign DNA (i.e. it restricts the ability of foreign DNA to infect the bacterial cell).

Function:

The cell guards against cutting its own DNA by adding a methyl group to one of the bases in all of the recognition sequences on each new DNA strand as soon as it is synthesized. This does not interfere with base pairing or gene function, but the restriction enzyme cannot act on the methylated sequence.

Mechanism of action: Each RE produces cleavage of both strands of DNA at specific nucleotide sequences formed of four to eight nucleotides (recognition sequences) in the form of a palindrome (symmetrical inverted repeat = a sequence of duplex DNA that is the same when the two strands are read in opposite directions).

Steps of Recombinant DNA technology

1- First step is cutting the DNA with restriction
Cuts are made in the plasmid DNA and cutting ends are created on both the sides of the gene segment by restriction endonucleases. The cutting of DNA results in: A-Sticky ends (overlapping or cohesive): They allow any two DNA fragments produced by the same restriction enzyme to form complementary base pairs. B-Blunt ends (cut along the axis of symmetry)

endonucleases:-

2- The cut ends can then be joined together or ligated by DNA ligase :

The desired gene segment is then inserted into the cut in the vector and treated with ligase. The cut ends of both a specific gene (to be amplified) and DNA of a vector are ligated to form a recombinant DNA molecule. Blunt ended DNA can also be joined together by DNA ligase but the reaction is much less frequent. This fusion of two segments takes place despite their different origins = chimera plasmid = chimeric DNA molecule. This modified plasmid is then either introduced into the original or in any other new strain of bacterium.

Recombinant DNA technology

Recombinant DNA technology

The resultant DNA is thus equivalent to the plasmid + an additional piece of genetic information.

The newly joined material = 'recombinant DNA'. Thus the host bacterium will follow the instructions of foreign DNA just as if it were its own.

The bacterium multiplies and its progeny cells continue to multiply normally, reproducing among their own genes the foreign one. The repeated divisions of the bacteria with new gene results in a colony of identical cells known as a clone.
The process of getting many identical copies of genetic material from one bacterium through multiplication of bacteria is known as cloning.

DNA amplification techniques

By Dr. Dalia Shaalan Lecturer of Biochemistry and Molecular Biology

DNA amplification techniques

1.

DNA amplification techniques include: DNA cloning : in vivo amplification technique.

2.

Polymerase chain reaction (PCR): in vitro amplification technique

I- DNA cloning
A clone refers to multiple identical copies produced from a single origin. There are two procedures of cloning: A- Genomic cloning. B- cDNA cloning .

Cloning a gene or a cDNA gives us large numbers of copies of a piece of DNA.

I- DNA cloning
A- Genomic cloning.
A genomic clone provides a piece of DNA identical in base sequence to the corresponding stretch of DNA in the cell.

B- cDNA cloning
The cDNA cloning is the isolation of a human cDNA (i.e. complementary DNA). It is the double-stranded DNA copy of a human mRNA.

The genomic DNA contains introns

Genomic clones are required for studies on gene structure.

cDNA does not.

cDNA clones are required when the aim is to produce proteins such as human insulin and growth hormone.

I- DNA cloning
Importance: This is necessary since many of the techniques used require visualization or quantification of the DNA and this requires many copies of the molecule for detection to be possible.
Cloning provides a mean of both separating and amplifying each piece of DNA. Aim of cloning: It makes a single piece of DNA to enter a bacterial cell and to be replicated in that cell. The bacterial cell will grow into a colony containing large numbers of identical cells.

Example: A single E. coli cell will produce ~107 cells in a single colony when grown on an agar plate overnight.

PCR has been established as one of the most widely used molecular biological techniques.

II- Polymerase chain reaction PCR

Advantages: The technique is a rapid, inexpensive and simple method for the amplification of very small amounts of DNA into much larger quantities.

It has become a routine method for amplifying specific sequences of DNA. It is used routinely in the diagnosis of a variety of genetic disorders.

II- PCR

1- DNA fingerprinting in the forensic laboratory: The PCR allows the DNA in a single cell, hair follicle, or sperm to be amplified and analyzed. 2- Detection of infectious agents, especially latent viruses: It can be used to pick out and amplify DNA sequences that are unique to invading organisms, and enable their identification (e.g. HCV and AIDS). 3- Prenatal genetic diagnosis. 4- Detection of allelic polymorphisms and molecular analysis of diseases. 5- Providing precise tissue typing for transplants. 6- Study of evolution, using DNA from archeological samples

Applications of PCR:

PCR

Molecular analysis of diseases

A- Detection of normal gene variations ( normal polymorphism) B- Detection of gene variations causing disease (abnormal polymorphism = Mutation) C- Prenatal diagnosis.

Molecular analysis of diseases

Types of polymorphisms
A- Restriction fragment length polymorphism (RFLP): It is an inherited difference in the pattern of restriction due to change (mutation) in a restriction enzyme cleavage site (palindrome).

Normal RFLPs have been found within known gene loci in sequences that have no known function.
When diseases caused from RFLP, they result from single-base changes (sickle cell disease) or from deletions or insertions of DNA into a coding restriction fragment (e.g. the thalassemias). Thus RFLPs may disrupt the function of the gene or may have no biologic effects.

Molecular analysis of diseases

Types of polymorphisms
B-Variable numbers of tandem repeat (VNTRs): Also referred as minisatellite. They consist of units of short nucleotide sequence (10-60 bp) that are tandemly repeated. VNTRs are common type of insertion that result in a RFLP. The VNTRs can be inherited and so they can be associated with a disease in a family or can be unique to an individual and serve as a molecular fingerprint of that person.

Molecular analysis of diseases

Types of polymorphisms
C- Microsatellite DNA polymorphisms: Also referred as Simple Sequence Repeats (SSRs) or short tandem repeats (STRs). They are short (2-6 bp) inherited, tandem repeat units of DNA occur about 50,000100,000 times in the human genome. Because they occur more frequently, they are replacing RFLP as the marker loci for various genome searches.

Molecular analysis of diseases

A- Detection of normal gene variations (normal polymorphism)
Individual variations of DNA sequence occur approximately once in every 500 nucleotides or about 107 times per genome. In healthy people, these alterations occur in non coding regions of DNA or at sites that cause no change in function of the encoded protein.

Molecular analysis of diseases

1- Point mutations The classic example is sickle cell disease. It is caused by mutation of a single base out of the 3x109 in the genome. T-to-A DNA substitution results in an A-to-U change in the mRNA corresponding to the sixth codon of the -globin gene. The altered codon specifies a different amino acid (valine rather than glutamic acid), and this causes a structural abnormality of the -globin molecule.

B- Detection of gene variations causing disease (abnormal polymorphism = Mutation)

Other point mutations in and around the -globin gene result in decreased or no production of -globin resulting in -Thalassemia.

Molecular analysis of diseases

2- Deletions, insertions and Rearrangements of DNA: The deletion of a critical piece of DNA, The rearrangement of DNA within a gene, or the insertion of a piece of DNA within a coding or regulatory region can cause changes in gene expression resulting in disease e.g -Thalassemia and -thalassemia.

Molecular analysis of diseases

C- Prenatal diagnosis:
If the genetic lesion is understood and a specific probe is available, prenatal diagnosis is possible. DNA from cells collected from amniotic fluid (or by chorionic villi biopsy) can be analyzed and the genetic defect is detected.

Some Applications and Techniques in Molecular Biology

By Dr. Dalia Shaalan Lecturer of Biochemistry and Molecular Biology

DNA Fingerprinting
It is a technique used mainly by forensic scientists to assist in the identification of individuals by their respective DNA profiles. It is used in, for example, parental testing and criminal investigation. VNTRs serve as a molecular fingerprint of a person because it is unique to every individual.

Gene therapy
It is a technique for correcting defective genes that are responsible for disease development. Diseases caused by defective gene product are treated by gene replacement therapy. The procedure is to clone a gene into a vector then it will be taken up and incorporated into the genome of a host cell. The introduced gene would begin to direct the expression of its protein product and this would correct the deficiency in the host cell. Gene therapy is directed towards the somatic cells and can not be passed on to the offspring.

Transgenic animals
A transgenic animal carries a foreign gene that has been inserted into its genome. The foreign gene is constructed using recombinant DNA methodology. A certain percentage of genes injected into a fertilized mouse ovum will be incorporated into the genome and found in both somatic and germ cells. The transgenic approach is used to correct a genetic deficiency in mice and their offsprings.

Transgenic animals
E.g. Fertilized ova obtained from mice with genetic hypogonadism were injected with DNA containing the coding sequence for the gonadotropin releasing hormone precursor protein. This gene will be expressed and regulated normally in the hypothalamus of a certain number of the resultant mice. These animals and their offspring showed no evidence of GnRH deficiency. In transgenic animals, one or more copies of a gene is added in the genome of the ovum and there is no way to control where the gene resides.

Gene knockout animals (Targeted gene disruption)

Gene knockout animals (usually knockout mouse) are made by creating a mutation that totally disrupts the function of a gene. Importance: To replace one of the two genes in an embryonic stem cell to create a heterozygous transgenic animal. The mating of two such animals will, by mendelian genetics, result in a homozygous mutation in 25% of offspring, thus a knockout mouse has had both alleles of a particular gene replaced with an inactive allele. Several hundred strains of mice with knockouts of specific genes have been developed. Knock out mice allow investigators to determine the role of a particular gene by observing the phenotype of individuals that lack the gene completely.

Special Techniques in Molecular biology

Two methods are used: 1- The Maxam-Gilbert sequencing method:

The ability to determine the sequence of DNA is fundamental to the success of much of molecular biology.

I- DNA sequencing

2- The Sanger method:

It is the original technique for sequencing and relies on the nucleotide-specific chemical cleavage of DNA and is not routinely used any more.

- It is the principal sequencing technique used and utilizes fluorescently labeled dideoxynucleotides (ddNTPs). - It has now been automated so that thousands of bases per day can be sequenced. - This development has made feasible the possibility of sequencing the whole human genome (for "human genome project").

Special Techniques in Molecular biology

II- Blotting Techniques
It is a technique used for visualization of specific DNA, RNA or protein molecules among thousands of different molecules. It includes: Southern blotting: for detection of DNA molecules Northern blotting: for detection of RNA molecules Western blotting: for detection of proteins

Blotting Techniques

Blotting Techniques
N.B: Probes:
A probe is a single strand of DNA that can hybridize with a complementary sequence on another single-stranded polynucleotide composed of DNA or RNA. The probe must be labeled by radioactive or fluorescent substance so that it can detect the specific complementary DNA or RNA sequence.

Blotting Techniques

It is the standard method for locating specific sequences in cloned DNA and also for identifying specific sequences within digests of total eukaryotic DNA. Firstly developed by Edward Southern in 1975, it takes the advantage of the fact that DNA fragments of different sizes can be separated in an electric field by agarose gel electrophoresis. The separated DNA fragments are then denatured with alkali, neutralized and then transferred (blotted) to nitrocellulose or nylon membrane. The attached DNA is then hybridized to a 32Plabelled cDNA or RNA probe that carries the complementary sequence of the gene of interest. The radiolabelled probe will bind to gene of interest and can be observed as an image on photographic film following autoradiography.

Southern blotting

Blotting Techniques
Northern blotting

It is similar to Southern blotting. RNA is electrophoresed, transferred and immobilized onto a nitrocellulose or nylon filter. The filter is then hybridized with a radiolabelled cDNA probe that is specific for a selected gene. Quantitation of mRNA expression is carried out by autoradiography followed by densitometry of the resultant photographic signal(s).

Western blotting

It is a method for transferring protein to a nitrocellulose filter, on which the protein can be detected by a suitable probe (e.g. an antibody).

Special Techniques in Molecular biology

III- DNA microarray (DNA chips)
This is used to screen thousands of genes simultaneously. cDNA probes for different genes are spotted or arrayed individually in high density on a glass slide or on nitrocellulose paper. Probes are hybridized to the microarray and binding is then quantified.

Biochemistry of Cancer
Cancer is a common cause of death allover the world. Humans of all ages develop cancer and a wide variety of organs are affected. Cancer cells are characterized by three properties: 1- Diminished control of cell division and growth. 2- Invasion of local tissues. 3- Spread or metastasis to other parts of the body and this is the major problem of the disease. Benign tumor cells have lost growth control but do not metastasize.

Biochemistry of Cancer
Carcinogenesis

Agents causing cancer can be classified into three groups: Radiant energy (such as ultraviolet rays, x-rays and -rays). Chemical compounds (such as polycyclic aromatic hydrocarbons, nitrosamines, alkylating agents, aflatoxin, arsenic, chromium, asbestos and cadmium). Viruses (such as papovavirus, adenovirus, herpesvirus and retrovirus). These agents damage or alter DNA so that cancer is truely a disease of the genome.

Genes involved in carcinogenesis:

Biochemistry of Cancer

A- Oncogenes They are genes capable of causing cancer. Normal cells contain potential precursors of oncogenes designated proto-oncogenes. Activation of these genes to oncogenes is achieved by the following mechanisms: 1- Promoter and enhancer insertion. 2- Chromosomal translocation. 3- Gene amplification. 4- Single-point mutation. Activated oncogenes affect cellular growth by disturbing normal cellular mechanisms of growth control and by acting as growth factors or receptors.

Biochemistry of Cancer
Genes involved in carcinogenesis (cont.)
B- Tumor suppressor genes When their activity is reduced, they result in uncontrolled cell growth. C- DNA repair genes Defective repair system is associated with carcinogenesis.

Biochemistry of Cancer

Many tumors are characterized by the presence of abnormal genes, enzymes, proteins and hormones, which can be used as markers for these tumors. The measurement of certain specific tumor markers in serum is now useful for diagnosis and follows up of certain tumors such as: 1- Cacinoembryonic antigen (CEA) in cancer colon, lung, breast and pancreas. 2- Alpha-feto protein (AFP) in cancer liver. 3- Prostatic specific antigen (PSA) in cancer prostate. 4- Human chorionic gonadotropin (hCG) in trophoblastic tumors; testicular tumors. 5- CA 19-9 in pancreatic cancer.

Tumor markers

Biochemistry of Cancer

It is programmed cell death (cell suicide). It is characterized by a decrease in cell volume, chromatin condensation with nuclear fragmentation, and cellular dispersion into fragmented apoptotic bodies without release of cellular material. There is activation of proapoptotic enzymes, called caspases. There are three different mechanisms by which a cell commits suicide by apoptosis by: 1- internal signals arising within the cell 2- death activators binding to receptors at the cell surface 3- dangerous reactive oxygen species

Apoptosis

Mechanisms of apoptosis