INTRODUCTION TO MOLECULAR MEDICINE

Dolores V. Viliran, M.D.

Department of Biochemistry

and Nutrition

INTRODUCTION TO MOLECULAR MEDICINE

DNA CLONINGPOLYMERASE CHAIN REACTION (PCR)DNA SEQUENCINGBLOT TECHNIQUESDNA FINGERPRINTINGRESTRICTION FRAGMENT LENGTH

POLYMORPHISM (RLFP)DNA CHIPSGENE THERAPY/TRANSGENESIS

Enzyme(s)Activity Comments

Restriction endonuclease

Recognizes specific nucleotide sequences; cleaves the DNA within or near the recognition sequences

Reverse transcriptase (RT)

RNA-dependent DNA polymerase; encoded by retrovirus

Used to convert mRNA into a complementary DNA (cDNA) copy for the purpose of cloning cDNAs

Rnase H Recognizes RNA-DNA duplexes; randomly cleaves the phosphodiester backbone of the RNA

Used primarily to cleave the mRNA strand that is annealed to the first strand of cDNA generated by reverse transcription

DNA polymerase

Synthesis of DNA Used during most procedures where DNA synthesis is required; also used in vitro mutagenesis.

Klenow DNA polymerase

Proteolytic fragment of DNA polymerase; lacks the 5’3’ exonuclease activity

Used to incorporate radioactive nucleotides DNA fragments generated by restriction enzymes; also can be used in place of DNA polymerase

DNA ligase Covalently attaches a free 5’ phosphate to a 3’ hydroxyl

Used in all procedures in which two molecules of DNA need to be covalently attached

Alkaline phosphatase

Removes phosphates from 5’ ends of DNA molecules

Used to allow 5’ ends to be radiolabeled with the -phosphate of ATP in the presence of polynucleotide kinase; also used to prevent self-ligation of restriction enzyme-digested plasmids and lambda vectors.

Polynucleotide kinase

Introduces -phosphate of ATP to 5’ ends of DNA

See above for alkaline phosphatase

Dnase 1 Ramdomly hydrolyzes the phosphodiester bonds of double-stranded DNA

Used in the identification of DNA regions that are bound by protein and thereby protected from Dnase 1 digestion; also used to identify transcriptionally active regions of chromatin, which are more susceptible to Dnase 1 digestion

S1 Nuclease Exonuclease that recognizes single-stranded regions of DNA

Used to remove regions of single-strandedness in DNA or RNA-DNA duplexes

Exonuclease III Exonuclease that removes nucleotides from the 3’ end of DNAs

Used to generate deletions in DNA for sequencing or to map functional domains of DNA duplexes

Terminal transferase

DNA polymerase that requires only a 3’-OH; lengthens 3’ ends with any dNTP

Used to introduce homopolymeric (same dNTP) “tails” onto the 3’ ends of DNA duplexes; also used to introduce radiolabeled nucleotides on the 3’ ends of DNA

T3, T7, and SP6

RNAPolymerasesb

RNA polymerase encoded by bacterial virus; recognizes specific nucleotide sequences for initiation of transcription

Used to synthesize RNA in vitro

Taq and Vent DNA polymerase

Thermostable DNA polymerases Used in PCR

Taq and Vent DNA ligases

Thermostable DNA ligases Used in LCR

Characteristics of Restriction Endonucleases:

Specific They act on nucleotide sequences that are

palindromic Some RE make staggered symmetrical cuts

away from the center of their recognition site within the DNA duplex = producing DNA with cohesive or sticky ends

Some RE make symmetrical cuts in the middle of their recognition site = producing fragments of DNA with blunt ends

Some RE cleave the DNA at a distance from the recognition sequence

CLONING

CLONINGProduction of large quantities of identical

DNA moleculesA vector is required to clone either cDNAs

or copies of genes.Classes of vectors:

PlasmidsLambdaYeast Artificial Chromosome (YAC)

vector

PLASMIDS

Circular DNAs found in bacteriaAutonomous replication separate

from the host genomeContain antibiotic resistance genes

that confer antibiotic resistance to bacteria

Limitation: Can only clone DNA less than 10,000 base pairs in length

LAMBDA

Derived from bacteriophage (bacterial virus)Bacteriophage can be classified as lysogenic or

lyticLysogenic- bacteriophage can integrate into the

host genomeLytic- infection followed by destruction of the

infected hostLimitation: Can carry fragments of DNA up to

25,000 base pairs in length

YEAST ARTIFICIAL CHROMOSOME (YAC)

Derived from yeastUse for cloning of DNA fragments up

to 500,000 base pairs

STEPS IN CLONING

Human chromosomal DNA is digested with restriction enzymes producing many DNA fragments of different sizes. Vector DNA is cleaved in the same way.

Vector DNA and human chromosome DNA is joined forming a recombinant DNA sealed with DNA ligase.

Recombinant DNA molecule is introduced into a host bacterium for replication.

Recombinant DNA is reproduced along with the host cell DNA.

cDNA CLONING

Production of library of cloned DNAs that represent all the mRNAs present in a particular tissue or cell.

Process begins with reverse transcription of the mRNA, followed by synthesis of the second strand of DNA and insertion of the dsDNA into a vector for cloning.

The vector is used to transform a host cell (e.g. E. coli)

Screening of cDNA clonesProbes: from nucleic acids or proteins

Expression cloning: with proteins, antibodies or biological assay

GENOMIC CLONING

Uses lambda-based vector systems, which are capable of carrying 15,000-25,000 bp of DNA

Use of a chimeric plasmid-lambda vector system termed “cosmid”:

Cosmid vectors contain only “cos” (cohesive) ends of the lambda genome (required for packaging the DNA into infectious virus particles

Digested with restriction enzymes to generate fragments in optimal size range for vector being utilized

Because some genes contain many more base pairs than can be inserted into a vector, overlapping clones are generated when DNA is only partially digested with restriction enzymes.

Partially digested DNA is size selected by a various techniques e.g. gel electrophoresis

Screening of genomic librariesNucleic acid probesProteins known to bind specific sequences of

DNA (e.g. transcription factors)

YEAST ARTIFICIAL CHROMOSOME (YAC) CLONING

YAC vectors allow cloning, within yeast cells, of fragments of genomic DNA that approach 500,000 base pairs.

Yeast vectors contain elements typical of yeast chromosomes Yeast centromere (CEN) Yeast telomere (TEL) Yeast autonomously replicating sequence (ARS) Genes that allow the selection of yeast cells that have taken up

the vector (e.g. URA3 gene invloved in uracil synthesis Bacterial replication origin: vector can be propagated in bacterial

cells Bacterial selectable marker: e.g. gene for ampicillin resistance

POLYMERASE CHAIN REACTION

Polymerase Chain Reaction

Xeroxing DNAMethod of amplifying a target sequence of

DNAAbility of DNA-copying enzyme to remain

stable at high temperatureCan only be done in test tubes

Polymerase Chain Reaction

Was first described by Dr. Karry Mullis , and it was this invention that he received a Nobel Prize in Medicine and Physiology in 1993.

In vitro enzymatic amplification of specific DNA sequence which involves three steps

which are repeated a number of times(cycle)

Three parts of the Polymerase Chain Reaction

1. DNA Denaturation

The double-stranded template DNA ( usually genomic DNA) is dissociated into single strands by heating the sample at 92 – 94o C (process of separation)

2. PRIMER Annealing

By lowering the temperature to 40 – 60oC, two oligonucleotide primers (typically 18-22 bases in length) can anneal to regions on the single DNA strand that flank the target DNA sequence to be amplified.

PRIMER Extensions

The 3’ end of the oligonucleotide primers are extended toward each other with newly synthesized DNA . This new DNA is complementary to target DNA sequences. To reduce non specific annealing of primers to DNA, this step is usually performed at an elevated temperature (e.g. 72oC using a thermostable DNA polymerase – typically Taq DNA polymerase from Thermus aquaticus)

TWO REQUIREMENTS OF THE POLYMERASE CHAIN REACTION

1. A supply of four nucleotide bases Adenine, Guanine , Cystine and Thymine

2. Primer

PCR TEST

- investigate a potentially defective DNA sequence

- diagnosis of diseases in which a specific mutational site is in question

- primers spanning the site are used-the amplified DNA can de sequenced or

otherwise compared with the wild type

Inherited Disorders Detected by PCRDISEASES AFFECTED GENE

Adenosine deaminase deficiency Adenosine deaminase

Lesch- Nyhan syndrome HGPRT

Alpha1- Antitrypsin Deficiency Alpha1- Antitrypsin

Fabry’s Disease Alpha-galactosidase

Cystic Fibrosis Cystic Fibrosis Transmembrane Conductance Protein(CFTR)

Gaucher’s Disease Glucocerebroside

Sandhoff-Jatzkewitz Disease Hexosaminidase A and B

Tay-sach’s Disease Hexosaminidase A

DISEASES AFFECTED GENE

Familial Hypercholesterolemia LDL receptor

Glucose-6-PhosphateDH Deficiency

Glucose-6-phosphate dehydrogenase

Maple Syrup urine disease Alpha keto acid decarboxylase

Phenylketonuria Phenylalanine Hydroxylase

Ornithine Transcarbamylase deficiency

Ornithine transcarbamylase

Retiniblastoma (Rb) Rb gene product

Sickle – cell anemia Point mutation in Beta globin gene resulting in improper folding of protein

Beta- Thalasssemia Mutations in Beta-globin gene that results in loss of synthesis of protein

DISEASES AFFECTED GENE

Hemophilia A Factor VIII

Hemophilia B Factor IX

Von Willebrand Disease Von Willebrand factor

PCR - SSCP

LIGASE CHAIN REACTION

Sanger’s Method of DNA Sequencing

Chain Termination Method

Based on DNA polymerase reaction Run four separate reactions Each reaction mixture contains dATP,

dGTP, dCTP and dTTP, one of which is P-32-labelled

Each reaction also contains a small amount of one dideoxynucleotide: either ddATP, ddGTP, ddCTP or ddTTP

Chain Termination Method

Most of the time, the polymerase uses normal nucleotides and DNA molecules grow normally

Occasionally, the polymerase uses a dideoxynucleotide, which adds to the chain and then prevents further growth in that molecule

Random insertion of dd-nucleotides leaves (optimally) at least a few chains terminated at every occurrence of a given nucleotide

Chain Termination Method

Run each reaction mixture on electrophoresis gel

Short fragments go to bottom, long fragments on top

Read the "sequence" from bottom of gel to top Convert this "sequence" to the complementary

sequence Now read from the other end and you have the

sequence you wanted - read 5' to 3'

Chemical Cleavage Method

Not used as frequently as Sanger's Start with ssDNA labelled with P-32 at one end Strand is cleaved by chemical reagents Assumption is that strands of all possible

lengths, each cleaved at just one of the occurrences of a given base, will be produced.

Fragments are electrophoresed and sequence is read

Chemical Cleavage Method

Four reactions are used G-specific cleavage with dimethyl sulfate,

followed by strand scission with piperidine G/A cleavage: depurination with mild acid,

followed by piperidine C/T cleavage: ring hydrolysis by hydrazine,

followed by piperidine C cleavage: same method (hydrazine and

piperidine), but high salt protects T residues

Chemical Cleavage Method

Reading the gels... It depends on which end of the ssDNA was

radioactively labelled! If the 5'-end was labelled, read the sequence

from bottom of gel to top (5' to 3') If the 3'-end was labelled, read the sequence

from top of gel to bottom (5' to 3') Note that the nucleotide closest to the P-32

will be missed in this procedure

Since the four dyes fluoresce at different wavelengths, a laser then reads the gel to determine the identity of each band according to the wavelengths at

which it fluoresces. The results are then depicted in the form of a chromatogram, which is a diagram of colored peaks that correspond to the

nucleotide in that location in the sequence (Russell, 2002).

Results from an automated sequence shown in the form of a chromatogram. The colors represent the four bases: yellow is C, pink is A, green is G and blue is T (Metzenberg).

The Fountain of Youth by Lucas Cranach

HYBRIDIZATION TECHNIQUESBlot Transfer procedures

Southern Blot

A test commonly used in molecular biology and genetics, the purpose of the test being to check for a match between DNA molecules.

Southern blot more formally called an DNA blot

Gene I

Genomic DNA

Restriction endonuclease

Gel Electrophoresis

Long DNA fragments

Short DNA fragments

Agarose gel

DNA fragments

-

+

DNA fragments

Agarose gel

-

+

1. Denature in alkali

2. Blot-transfer bake

ssDNA fragments

nitrocelluloseRadioactive RNA or denatured DNA containing sequences complementary to gene I (radioactive probe)

1. Hybridize nitrocellulose with radioactive probe

2. wash

Autoradiography

Photographic film

An example of a real Southern blot used to detect the presence of a gene that was transformed into a mixed cell population.

Northern Blot

A technique in molecular biology, used mainly to separate and identify pieces of RNA.

Northern blot more formally called an RNA blot.

Probe = cDNA

Preparation of a radioactive probe:

Purified mRNA

+

DNA primer

Reverse transcriptase(DNA Polymerase)

+ 32 P-dNTPs

DNA-RNA hybrid

1

RNase(destroy RNA)

DNA-RNA hybrid

Radioactive cDNA probe2

Gel electrophoresis of total mRNA (agarose)

weight

Paper towel

membrane

Gel

Wet paper towel

Transper of RNA from gel to membrane by

blotting

Total RNA distribution

Side view: RNA is retained by the membrane

Radio-active bands

(hybridized probe)

Radioactive RNA hybridizes only to its complementary sequence

Dry and expose to X-ray film

Hybridized RNA on membrane to specific DNA probe

Northern blot (X-ray film)

Pre mRNA

Partially spliced

Partially spliced

mRNA

an example of Northern Blot

Western Blot

A technique in molecular biology, used to separate and identify proteins.

Western blot more formally called a protein immunoblot.

SDS PolyacrylamideGel Electrophoresis

Protein Blot on Nitrocellulose

Label with specific antigen Detect Antibody

Reveal protein of interest

•DNA fingerprinting is a system identifying organisms, disease and paternity based on DNA analysis

•use to solve crimes by identifying the victim, criminal and other participants in a criminal act.

Procedure:

Performing a Southern Blot

Making a Radioactive Probe

Creating a Hybridization Reaction

VNTRs

Performing a Southern Blot

Making a Radioactive Probe

Introduce horizontal breaks along a strand, , add individual nucleotides to the nicked DNA, one of which, *C [light blue], is radioactive.

Add the DNA polymerase

DNA polymerase will become immediately attracted to the nicks in the DNA and attempt to repair the DNA, starting from the 5' end and moving toward the 3' end

The DNA polymerase begins repairing the nicked DNA. Whenever a G base is read in the lower strand, a radioactive *C [light blue] base is placed in the new strand

the nicked strand, as it is repaired by the DNA polymerase, is made radioactive by the inclusion of radioactive *C bases.

The nicked DNA is then heated, splitting the two strands of DNA apart

This creates single-stranded radioactive and non-radioactive pieces. The radioactive DNA, now called a probe [light blue], is ready for use.

Creating a Hybridization Reaction

Hybridization is the coming together, or binding, of two genetic sequences. The binding occurs because of the hydrogen bonds [pink] between base pairs.

DNA must first be denatured, usually by using heat or chemicals.

The denatured DNA is put into a plastic bag along with the probe and some saline liquid; the bag is then shaken to allow sloshing. If the probe finds a fit, it will bind to the DNA

The fit of the probe to the DNA does not have to be exact. Sequences of varying homology can stick to the DNA even if the fit is poor; the poorer the fit, the fewer the hydrogen bonds between the probe [light blue] and the denatured DNA. The ability of low-homology probes to still bind to DNA can be manipulated through varying the temperature of the hybridization reaction environment, or by varying the amount of salt in the sloshing mixture.

VNTRs

•Every strand of DNA has exons and introns•introns contain repeated sequences of base pairs called Variable Number Tandem Repeats (VNTRs) . (twenty to one hundred base pairs) •Every human being has some VNTRs•To determine if a person has a particular VNTR, a Southern Blot is performed, and then the Southern Blot is probed, through a hybridization reaction, with a radioactive version of the VNTR in question•The pattern which results from this process is what is often referred to as a DNA fingerprint• A given person's VNTRs come from the genetic information donated by his or her parents; he or she could have VNTRs inherited from his or her mother or father, or a combination, but never a VNTR either of his or her parents do not have

Because VNTR patterns are inherited genetically, a given person's VNTR pattern is more or less unique. The more VNTR probes used to analyze a person's VNTR pattern, the more distinctive and individualized that pattern, or DNA fingerprint, will be.

Applications of DNA fingerprinting

Paternity and Maternity

Criminal Identification and Forensics

Personal Identification

Restriction Map

Restriction fragment length polymorphism (RFLP)

Restriction fragment length polymorphism is the identification of specific restriction enzymes that reveal a pattern difference between the DNA cut DNA at specific 4-6 bp recognition sites

Sample DNA is cut with one or more RE’s and resulting fragments are separated according to molecular size using gel electrophoresis

Differences result from base substitutions, additions, deletions or sequence rearrangements within RE recognition sequences

Presence and absence of fragments resulting from changes in recognition sites are used identifying species or populations.

Applications

This can be used to genetically tell individuals apart.

It can show the genetic relationship between individuals, because children inherit genetic elements from their parents.

It is used to determine the relationships among species.

Restriction enzymes cut DNA at precise points producing

A collection of DNA fragments of precisely defined length.These can be separated by electrophoresis with smaller fragments migrating farther than the larger fragments.One or more fragments can be visualized with a “probe” – molecule of single stranded DNA that is

Complementary to a run of nucleotides in one or more of the restriction fragments and is

Radioactive (or fluorescent)

Cases where RFLP is use

Sickle-cell disease The only difference between thetwo genes is the substitution of a Tor an A in the middle position ofcodon 6

A change of a single nucleotideproduced the RFLP

This is a very common cause ofRFLPs also called as a singleNucleotide polymorphisms orSNPs

Screening for Sickle-cell anemia

DNA Microchips

•a mutation - in a particular gene's DNA often results in a certain disease

•The DNA microchip is a revolutionary new tool used to identify mutations in genes

•The chip, which consists of a small glass plate encased in plastic, is manufactured somewhat like a computer microchip

•On the surface, each chip contains thousands of short, synthetic, single-stranded DNA sequences, which together, add up to the normal gene in question.

Use of DNA Microchips

Research tool

•As we gain more insight into the mutations that underly various diseases

• DNA chips can help assess individual risks for developing different cancers as well as heart disease, diabetes and other diseases.

Procedure

determine whether an individual possesses a mutation for certain genes, a scientist first obtains a sample of DNA from the patient's blood as well as a control sample

denatures the DNA in the samples - a process that separates the two complementary strands of DNA into single-stranded molecules.

cut the long strands of DNA into smaller, more manageable fragments and then to label each fragment by attaching a fluorescent dye. The individual's DNA is labeled with green dye and the control - or normal - DNA is labeled with red dye. Both sets of labeled DNA are then inserted into the chip and allowed to hybridize

If the individual does not have a mutation for the gene, both the red and green samples will bind to the sequences on the chip.

If the individual does possess a mutation, the individual's DNA will not bind properly in the region where the mutation is located. The scientist can then examine this area more closely to confirm that a mutation is present.

DNA CHIPS - Microarray technology

This technology promises to monitor the whole genome on a single chip so that researchers can have a better picture of the interactions among thousands of genes simultaneously.

Principle:Base-pairing (i.e., A-T and G-C for DNA;

A-U and G-C for RNA) or hybridization is the underlining principle of DNA microarray.

The sample spot sizes in microarray are typically less than 200 microns in diameter and these arrays usually contains thousands of spots.

Application

Identification of sequence (gene/ gene mutation) determination of expression level (abundance) of genes.The microarray (DNA chip) technology is having a

significant impact on genomics study Many fields, including drug discovery and toxicological research, will certainly benefit from the use of DNA microarray technology.

Disease diagnosis Drug discovery: Pharmacogenomics

Gene therapy is a technique for correcting defective genes responsible for disease development.

Gene therapy is designed to introduce genetic material into cells to compensate for abnormal genes or to make a beneficial protein. If a mutated gene causes a necessary protein to be faulty or missing, gene therapy may be able to introduce a normal copy of the gene to restore the function of the protein.

Several approaches for correcting faulty genes

A normal gene may be inserted into a nonspecific location within the genome to replace a nonfunctional gene. This approach is most common.An abnormal gene could be swapped for a normal gene through homologous recombination.The abnormal gene could be repaired through selective reverse mutation, which returns the gene to its normal function.The regulation (the degree to which a gene is turned on or off) of a particular gene could be altered.

The most common techniques utilized in gene therapy studies is the introduction of the corrected gene into bone marrow cells, skin fibroblasts or hepatocytes. The vectors most commonly utilized are derived from retroviruses and utilize only the transcriptional promoter regions of these viruses (the LTRs) to drive expression of the gene of interest. The advantage of retroviral-based vector systems is that expression occurs in most cell types. A number of human inherited disorders have been corrected in cultured cells and several diseases (e.g. malignant melanoma and severe combined immunodeficiency disease, SCID) are currently being treated by gene therapy techniques indicating that gene therapy is likely to be a powerful therapeutic technique against a host of diseases in coming years.

A gene that is inserted directly into a cell usually does not function. Instead, a carrier called a vector is genetically engineered to deliver the gene. Certain viruses are often used as vectors because they can deliver the new gene by infecting the cell. The viruses are modified so they can't cause disease when used in people. Some types of virus, such as retroviruses, integrate their genetic material (including the new gene) into a chromosome in the human cell. Other viruses, such as adenoviruses, introduce their DNA into the nucleus of the cell, but the DNA is not integrated into a chromosome.

The vector can be injected or given intravenously (by IV) directly into a specific tissue in the body, where it is taken up by individual cells. Alternately, a sample of the patient's cells can be removed and exposed to the vector in a laboratory setting. The cellscontaining the vector are then returned to the patient. If the treatment is successful, the new gene delivered by the vector will make a functioning protein.

new gene is injected into an adenovirus vector, which is used to introduce the modified DNA into a human cell. If the treatment is

successful, the new gene will make a functional protein.

Some of the different types of viruses used as gene therapy vectors:

Retroviruses

Adenoviruses

Adeno-associated viruses

Herpes simplex viruses

Current gene therapy research has focused on treating individuals by targeting the therapy to body cells such as bone marrow or blood cells. This type of gene therapy cannot be passed on to a person’s children. Gene therapy could be targeted to egg and sperm cells (germ cells), however, which would allow the inserted gene to be passed on to future generations. This approach is known as germline gene therapy.(TRANSGENESIS)

Steps Involved in a Human Germline Gene Therapy Protocol

1. Isolation of totipotent embryonic cells at an undifferentiated stage

2. Determination of the genetic state of the embryo.3. Expansion of embryonic stem cells in culture. 4. Transfer of genetic material into embryonic cells 5. Selection of cells which have stably taken up the

transfected gene6. Targeted gene replacement 7. Marker removal. 8. Confirming genomic integrity 9. Nuclear transfer 10. Reimplantation into the mother

MAKING RECOMBINANT DNA

Uses

Medicine

- Genetic engineering is able to treat many illnesses and conditions with the human body which were previously much more harmful. Many medicines and treatments are available only because of this technology

Agriculture

-Much of the food we eat are in some way connected to genetic engineering. In fact, about 60 percent of our food has some sort of biotechnology in it. By taking traits from one organism and putting it into a food, the food can be altered in many ways, like having it last longer, taste better, and grow faster and larger. It can also be designed to be more immune to certain diseases.

Industry - The ability of bacteria to produce chemicals can be used in other areas as well such as in the cheese industry. Also, genetically related advances and business is booming.

Terrorism Former Soviet Union used traits in many organisms to make biological weapons and viruses never before heard of. One scenario is that by using recombinant DNA methods, they might have taken the lethality of Ebola, combined it with the worst part of anthrax, and to top it off, made it extremely contagious by including parts of the smallpox virus.

DNA RECOMBINANT TECHNOLOGY SYNTHESIS OF HUMAN INSULIN

What are Biotechnology Drugs?

Drugs produced using living organisms such as yeast , bacteria , or mammalian cells.

Examples: Cytokines Human Growth Hormone ( hGH) Insulin Interferon Factor Erythropoietin Monoclonal Antibodies

Approved Biotechnology Drugs 1999

1. Activase (Alteplase recombinant): treatment of acute myocardial infarction , acute massive pulmonary embolism, acute ischemic stroke within the first hours of symptom onset.

2. Alphanate ( human antihemophilic factor): treatment of hemophilia or acquired Factor VII deficiency.

3. Humalog ( recombinant insulin) : treatment of diabetes.

4. Geref: treatment of growth hormone deficiency in children with growth failure.

5. GenoTropin: treatment of growth hormone deficiency in children; growth deficiency in adults.

6.Vistide(cidofovir injection)/ Vitravene (fomivirsen sodium, injectable): treatment of (CMV) retinitis in AIDS.

7.Zenapax (Daclizumab): humanized monoclonal antibody for prevention of kidney transplant rejection.

(The medicines and vaccines included in this list are produced and/or developed by companies involved in recombinant DNA research or other biotechnology applications.).

Thank you!!!!