Techniques in Molecular BiologyNaglaa Alhusseini

RECOMBINANT DNA TECHNOLOGY

Definition: Recombinant DNA technology is genetic engineering

which effects artificial modifications of genetic constitution of a living cell by introduction of foreign DNA through experimental techniques.

The technique involves:1. Splicing of DNA by restriction endonucleases

2. Preparation of chimeric molecules

3. Cloning of large number of identical target DNA molecules.

Tools of Recombinant DNA technology:

Enzymes : Restriction endonucleases- DNA polymerases- DNA ligase – Reverse transcriptase

Vectors or vehicle DNA : DNA which act as a carrier which includes : Bacterial plasmid – Bacteriophages – Cosmids

Passenger DNA( foreign DNA ): DNA which insert into the vector DNA , they are; Synthetic DNA- Random DNA – cDNA (complementary DNA)

Hosts :

1. Restriction Endonucleases:

These are bacterial enzymes which have the ability to cleave DNA molecules at certain sites with high specific sequence at the cleavage site.

It is called endonucleases because they cut the polynucleotide in the middle.

Most regions that are recognized by the enzymes are Palindromes, the two strands have the same

sequence in the 5` to 3` direction.

2-Reverse transcriptase:

It is an enzyme derived from viruses, oncogenic (tumor producing) viruses or retroviruses.

Its name derived from its action of reversing the usual procedure i.e. instead of producing mRNA from DNA as a usual process, reverse transcriptase enzyme reverse the procedure producing cDNA from RNA, while the last act as a template.

3-DNA ligase: DNA ligase is an important tool for joining the

sticky ends in the preparation of recombinant DNA.

4-DNA polymerases: They make complementary copies of DNA

templates . there are several types of DNA polymerases . Examples, DNA polymerase I and Taq polymerase (thermostable DNA polymerase)

2. Vectors:

Plasmids It is a segment of DNA to which the fragment

of DNA to be cloned is attached. Plasmids are the most popular vectors . It occurs in certain microorganisms (e.g.

bacteria). It is often contains antibiotic resistance genes;

a method of selection of cells containing - recombinant DNA molecule (e.g. growth in presence of antibiotic). Only the bacteria containing the plasmids will grow.

It is extra chromosomal, intra cytoplasmic circular DNA found in bacteria.

It is much smaller than bacteria DNA produced few thousand of nucleotide pairs, so it easy to separate them from each other.

Plasmids multiply independently from bacterial host cells (autonomous replication).

Bacteriophages: They are usually having linear DNA molecule

into which foreign DNA can be inserted at several restriction enzyme sites.

They can accept DNA fragment 10-20kb long( one kb =1000nucleotides base sequence)

Cosmids They are plasmid that also contain some

portion of bacteriophage DNA They are offer advantages of both plasmid and

bacteriophage vectors, as can be used to clone DNA insert as long as 45kb.

Passenger DNA(foreign DNA)

Synthetic DNA: Synthetic DNA can be produced purely by chemical means They are short segments of DNA(10-15 nucleotides)

Random DNA: It is random pieces produced by a shot gun experiment

using restriction endonuclease enzymes

cDNA (complementary DNA): Reverse trascriptase ( RNA dependant DNA polymerase)

makes complementary copies (cDNA) of mRNA templates The cDNA does not contain introns which are present in

the genomic DNA sequence and removed from mRNA during splicing process( post-transcription modification ) described before.

General Applications of Recombinant DNA Technology:

1. Manufacture of proteins / hormones : Proteins: such as plasminogen, interferon and blood

clotting factors. Hormones: e.g. insulin and growth hormones…….

2. AIDS test : by using recombinant DNA technology , the diagnosis of diseases like AIDS by laboratory has become simple and rapid

3. Diagnosis of molecular diseases: for example Sickle cell anemia , thalassaemias , familial hypercholesterolemia , cystic fibrosis …..etc

4. Prenatal diagnosis of the genetic lesions5. Gene therapy

6. Applications in agriculture: Genetically engineered plants have been developed to resist diseases, good quality of food and increased yield in crops could be possible by applications of this technique.

7. Industrial applications: Recombinant DNA technology used for synthesis of enzymes which are used to produce sugar, cheese and detergents. Beside certain proteins products used as food additives, to increase the nutritive value beside flavor.

8. Applications in forensic medicine : genetic engineering have greatly helped to identify criminals and settle the doubtful of parenthood of children

9. Transgenesis :The somatic gene replacement therapy will not pass to the offspring , transgenesis refers to the transfer of gene into fertilized ovum , which will be found in somatic cells as well as germ cells and passed on to the successive generation

TECHNIQUES IN MOLECULAR BIOLOGY

1. Isolation of DNA2. Electrophoresis of DNA3. Hybridization and Blotting

Techniques4. Techniques for DNA

Amplification 5. Restriction Fragment Length

Polymorphisms (RFLP)6. DNA Finger Printing (DNA

Typing):

Techniques for DNA Amplification

I-DNA cloning (Gene Cloning) DNA cloning allows production for large number of

identical DNA (genes) which can be used for another purpose.

It is in vivo DNA amplification

The process consists of:-

1. Obtaining the gene.

2. Insertion of the gene into a vector (Plasmid or phages).

3. Multiplication of the vector in a host cell (bacteria).

4. Separation of the vector from the host cell.

5. Separation of the gene from the vector.

6. Hybridization i.e. specific fragment of cloned gene can be identified with complementary probe.

1) Obtaining copies of the gene of interest:

a) Restriction enzymes (large No. of genes), these enzymes are isolated from microorganism's cellular genome and cut DNA only at the site of the nucleotide sequence recognized by these enzymes.

b) From mRNA by the reverse

transcriptase enzyme, where mRNA in a reverse process act as a template to form a single strand of DNA (cDNA) that completed to form double strand DNA.

2-Digestion by restriction enzyme:

1. The DNA to be cloned is cleaved with a restriction endonuclease:- Restriction endonuclease recognizes short sequences in DNA and cleaves both strands within this region. Most of the DNA regions recognized by these enzymes are palindromes, i.e. the two strands of DNA have the same base sequence in the 5` - 3` direction.

2. Cleavage of vector DNA with the same restriction enzyme that is used for cleaving the target DNA

3-Production of recombinant DNA molecule :

The plasmid and foreign DNA (inserted DNA) both cleaved by the same restriction enzyme are mixed together and treated with ligase enzyme.

DNA ligase enzyme used to join (re-annealing) the fragment to the DNA vector produced chimeric plasmid contain the DNA insert into the plasmid (recombinant DNA).

4-Multiplication of the vector in a host cell

The chimeric plasmid are introduced into bacterial host cell the process of introducing foreign DNA into bacteria is termed “transformation”, Alternatively, introducing foreign (recombinant DNA) into viral genome is termed “Transfection” ,i.e. the virus is infected and then infects the host cells, introducing the recombinant DNA into the host cell genome. Only 5% of bacteria colonies contain the desired vector, so we have to select the desired colonies

The bacterial host cell containing the recombinant vector can be selected if the vector contains an antibiotic resistance gene. Bacteria without vector die in the presence of antibiotic.

5- Isolation of cloned foreign DNA or its protein product: Cells containing an appropriate chimeric plasmid

are cultured.To obtain large quantities of the foreign DNA, the plasmid are isolated from host cells (the bacteria are lysed and the hybrid plasmids are isolated) then treated with the restriction enzyme to release the foreign DNA, the plasmids are cut with the same restriction enzyme releasing many copies of the DNA of interest.

If the host cells are grown under conditions that permit the production of protein produced from target DNA, then the protein of interest can be isolated.

6-Detection of either cloning DNA or RNA:

By using labeled polynucleotide which complementary to the target DNA or RNA.

Importance of Gene Cloning 1. It can be used for production of viral coat protein,

which could be used for immunization of human against e.g. Rabis or hepatitis (B. or C.), used for synthesis of vaccines.

2. Can be used for the production of interferon with a value in the treatment of viral infection and possibly in cancer.

3. It explain the molecular bases of genetic diseases and treated it by gene replacement (Gene therapy) i.e. correction of genetic defect.

4. It is used in the synthesis of large quantities of human proteins (Multiple copies of the normal genes) e.g. insulin and GH.

5. It is used pharmaceutically in the production of large quantities of protein, i.e. certain antibodies in pure form which are used for diagnostic test e.g. pregnancy test or hormonal assays.

II-Polymerase Chain Reaction (PCR)

PCR is defined as an in vitro DNA amplification procedure in which millions of copies of a particular sequence of DNA can be produced within a few hours. It is like Xerox machine for gene copying.

This is an amplification reaction in which a small amount of a DNA template is amplified to provide enough copies (up to 100,000 fold) to perform analysis (prenatal diagnosis, detection of an infectious organisms or the presence of a mutated oncogene).

It is an extremely sensitive technique than does cloning technique.

Karry Mullis invented this method in 1989, who was awarded Nobel Prize in 1993.

Material required: Target DNA may be chosen from any source such

as leukocytes, tissues and single cells Two synthetic oligonucleotide primers: These

primers are complementary to the end of each strand of target DNA to be amplified. The selection of the primer requires the knowledge of the flanking sequences of the gene of interest. Two primers of about 20-30 nucleotides with complementary sequence of the flanking region can be synthesized.

Heat stable DNA polymerase: This enzyme is derived from bacteria Thermus acquaticus that are found in hot springs. Therefore the enzyme is not denatured at high temperature. This polymerase is not denatured even at temperature around 95oC.

Heat-stable polymerase is vitalHeat-stable polymerase is vitalto the ease of the processto the ease of the process

How is this different from cloning?How is this different from cloning?

In vitro amplification (in a test tube)In vitro amplification (in a test tube) Enzymatic: Taq polymeraseEnzymatic: Taq polymerase – – Temperature-resistant DNATemperature-resistant DNA polymerase ( Thermus aquaticus)polymerase ( Thermus aquaticus) Heat resistantHeat resistant Best for <2 kb targetBest for <2 kb target

Problems with TaqProblems with Taq

Taq DNA polymerase - thermostableTaq DNA polymerase - thermostable Lack of 3′-5′ exonuclease – proofreadingLack of 3′-5′ exonuclease – proofreading ► ►Error rate = 2 × 10-4 nucleotdes/cycleError rate = 2 × 10-4 nucleotdes/cycle Newer polymerases have high fidelityNewer polymerases have high fidelity High fidelity polymerase - HiFi TaqHigh fidelity polymerase - HiFi Taq

Technique steps: Denaturation: mixture heated to up 95°C for

15 seconds to 2 minutes to separate the two DNA strands . The two separate strands serve as complementary strand for DNA elongation reaction.

Annealing, The reaction mixture is cooled to about 50 -65oC for about 1-3 minutes. The temperature chosen for cooling is usually about 2-3 below Tm.(melting temperature).

Extension. The elongation of DNA from the site of annealing of primers is catalyzed by Taq DNA polymerase. The polymerase reaction is allowed to take place at 72°C for 30 seconds in the presence of dNTPs (all four deoxy ribonucleotide triphosphates).

Identification of the PCR products:

After the amplification procedure, DNA electrophoresis and Southern blot analysis with a suitable probe shows the presence of the DNA in the sample tissue

RT-PCR (Reverse Transcription PCR) refers to utilization of mRNA for the formation of target DNA using reverse transcriptase enzyme.

Various Types of PCR

Multiplex-PCR: consists of multiple primer sets within a single PCR mixture to produce amplicons of varying sizes that are specific to different DNA sequences. By targeting multiple genes at once, additional information may be gained from a single test run that otherwise would require several times the reagents and more time to perform. Annealing temperatures for each of the primer sets must be optimized to work correctly within a single reaction, and amplicon sizes, i.e., their base pair length, should be different enough to form distinct bands when visualized by gel electrophoresis.

Simultaneously modification of more than

one locus in the same reaction

Rapid and convenient – screening

Included different set of primers

Nested PCR: increases the specificity of DNA

amplification, by reducing background due to non-specific amplification of DNA. Two sets (instead of one pair) of primers are used in two successive PCRs. In the first reaction, one pair of primers is used to generate DNA products, which besides the intended target, may still consist of non-specifically amplified DNA fragments. The product(s) are then used in a second PCR with a set of primers whose binding sites are completely or partially different from and located 3' of each of the primers used in the first reaction. Nested PCR is often more successful in specifically amplifying long DNA fragments than conventional PCR, but it requires more detailed knowledge of the target sequences.

Quantitative PCR (Q-PCR): used to measure the quantity of a PCR product (commonly in real-time) RT-PCR. It quantitatively measures starting amounts of DNA, cDNA or RNA. Q-PCR is commonly used to determine whether a DNA sequence is present in a sample and the number of its copies in the sample. Quantitative real-time PCR has a very high degree of precision. QRT-PCR methods use fluorescent dyes, such as Sybr Green, EvaGreen or fluorophore-containing DNA probes, such as TaqMan, to measure the amount of amplified product in real time. It is also sometimes abbreviated to RT-PCR (Real Time PCR) or RQ-PCR. QRT-PCR or RTQ-PCR are more appropriate contractions, since RT-PCR commonly refers to reverse transcription PCR (see below), often used in conjunction with Q-PCR.

In situ PCRIn situ PCR

The advantages of real time PCR are rapid, very sensitive, reproducible , reduced risk of contamination (sealed reactions), easy to perform and allow for quantitation of result using software driven operation.

Reverse Transcription PCR (RT-PCR): for amplifying DNA from RNA. Reverse transcriptase reverse transcribes RNA into cDNA, which is then amplified by PCR. RT-PCR is widely used in expression profiling, to determine the expression of a gene or to identify the sequence of an RNA transcript, including transcription start and termination sites. If the genomic DNA sequence of a gene is known, RT-PCR can be used to map the location of exons and introns in the gene. The 5' end of a gene (corresponding to the transcription start site) is typically identified by RACE-PCR (Rapid Amplification of cDNA Ends)

Clinical Applications of PCR 1. Diagnosis of bacterial and viral diseases: In early phases of tuberculosis, the sputum may

contain only very few tubercle bacilli, so that usual acid fast staining may be negative. But PCR could detect even one bacillus present in the specimen.

Any other bacterial infection could also be detected similarly.

The specific nucleotide sequences of the bacilli are amplified by PCR and then detected by Southern blot analysis.

If reverse PCR is done, living organisms could be detected. This technique is widely used in the diagnosis of viral infections like Hepatitis C, Cytomegalovirus and HIV.

3. Diagnosis of genetic disorders: The PCR technology has been widely used to

amplify the gene segments that contain known mutations for diagnosis of inherited diseases such as sickle cell anemia, beta thalassemia, cystic fibrosis,

4. PCR is especially useful for prenatal diagnosis of inherited diseases, where cells obtained from fetus by amniocentesis are very few.

5. Cancer detection: PCR is widely used to monitor residual abnormal cells present in treated patients. Similarly identification of mutations in oncosuppressor genes (e.g. p53) and retinoblastoma gene, etc. can help identify individuals at high risk of cancer (oncogenes and oncosuppressor genes).

2. Medico-legal cases: PCR allows the DNA in a single cell or in a hair follicle to be analyzed. The restriction analysis of DNA from the hair follicle from the crime scene is studied after PCR amplification.

This pattern is then compared with the restriction analysis of DNA samples obtained from various suspects; the culprit’s sample will perfectly match with that of PCR amplified sample. The restriction analysis pattern of DNA of one individual will be very specific (DNA finger printing); but the pattern will be different from person to person. This is highly useful in forensic medicine to identify the criminal.

6- Quantification of gene expression (mRNA template is first reverse transcribed into a cDNA equivalent before amplification (RT-PCR).

7- Tissue typing for transplanting, by PCR and detection of genetic variants, especially of MHC (Major histocompatibility complex).

8. Fossil studies: DNA isolated from fossils and amplified by PCR is used to study evolution by comparing the sequences in the extinct and living organisms.