Nucleic acid Diagnostics and Removal Technics from

BiopharmaceuticalsBy: Elaheh Entezar-Almahdi, Pharm.D

Ph.D student of Pharmaceutics

Some References:

Recombinant DNA Cloning Procedure

Plasmid Cloning by Restriction Enzyme

Transformation

• Bacterial cells are suspended in a solution of calcium chloride at 0 °C.• Positively charged calcium. ions stabilize the negative charges of

phosphates on the membrane.• The low temperature freezes the cell membrane making it more

rigid.• This stabilizes the cell membrane both physically and

chemically.• Next the plasmid DNA is introduced into the solution.

DNA Extraction

AccuPrep® Nano-Plus Plasmid mini Extraction kit

Gel Electrophoresis

Gel Electrophoresis (Cont’d)

• A: 1kb DNA Ladder• B: pUMVC3-hIL12 (lower

part)• C: pUMVC3-hIL12 (upper

part)• D: pRL-CMV-Luc (lower

part)• E: pRL-CMV-Luc (upper

part)• F: pUMVC3-hIL12 (lower

part) (second elution)All Rights Reserved for Entezar-Almahdi et al,.

Polymerase Chain Reaction (PCR)• PCR is a technique which is used to amplify the number of copies

of a specific region of DNA, in order to produce enough DNA to be adequately tested.

• The purpose of a PCR is to make a huge number of copies of a gene. As a result, it now becomes possible to analyze and characterize DNA fragments found in minute quantities in places like a drop of blood at a crime scene or a cell from an extinct dinosaur.

• PCR is valuable to researchers because it allows them to multiply unique regions of DNA so they can be detected in large genomes.

PCR (Cont’d)• When first developed, multiple cycles

of the PCR process were cumbersome for two reasons:

First, the DNA polymerases (Klenow fragment) available at the time were inactivated each time the temperature was raised to denature the template strand.

Second, three water baths at three different temperatures were necessary, which meant that constant human attention was required.

PCR (Cont’d)

Two developments were instrumental in the maturation of the PCR process.

• First was the purification of a heat-stable DNA polymerase (Taq DNA polymerase).

• The second development was the invention of a thermal cycler.

PCR (Cont’d)

RT PCR

Reverse transcription polymerase chain reaction (RT-PCR) is a variant of polymerase chain reaction (PCR), a laboratory technique commonly used in molecular biology to generate many copies of a DNA sequence, a process termed "amplification". In RT-PCR, however, an RNA strand is first reverse transcribed into its DNA complement (complementary DNA, or cDNA) using the enzyme reverse transcriptase, and the resulting cDNA is amplified using traditional PCR or real-time PCR. Reverse transcription PCR is not to be confused with real-time polymerase chain reaction (Q-PCR/qRT-PCR), which is also sometimes abbreviated as RT-PCR.

Real Time PCR

• For one or more specific sequences in a DNA sample, Real Time-PCR enables both detection and quantification.• This is a new approach compared to standard PCR, where the product of the

reaction is detected at its end. Two common methods for detection of products in real-time PCR are:

1. non-specific fluorescent dyes that intercalate with any double-stranded DNA,2. sequence-specific DNA probes consisting of oligonucleotides that are labeled

with a fluorescent reporter which permits detection only after hybridization of the probe with its complementary DNA target.

• Frequently, real-time PCR is combined with reverse transcription to quantify messenger RNA and Non-coding RNA in cells or tissues.

Real Time PCR

1. The PCR is prepared as usual, and the reporter probe is added.2. As the reaction commences, during the annealing stage of the PCR both probe and primers anneal to the DNA target.

3. Polymerisation of a new DNA strand is initiated from the primers, and once the polymerase reaches the probe, its

5'-3'-exonuclease degrades the probe, physically separating the fluorescent reporter from the quencher, resulting

in an increase in fluorescence.

4. Fluorescence is detected and measured in a real-time PCR machine, and its geometric increase corresponding to

exponential increase of the product is used to determine the threshold cycle (CT) in each reaction.

DNA Hybridization ProbeA general laboratory nucleic acid hybridization scheme is as follow;1. Bind single-stranded DNA (the target)

to a membrane support.2. Add single-stranded labeled DNA (the

probe) under appropriate conditions of temperature and ionic strength to promote base pairing between the probe and the target DNAs.

3. Wash the support to remove excess unbound labeled probe DNA.

4. Detect the hybrid sequences that form between the probe and target DNA.

Detection Methods

Nonradioactive Hybridization Procedure1. The biotin-labeled probe is hybridized to

the target DNA.2. Either avidin, a chicken egg white protein,

or streptavidin, a bacterial analogue of avidin, is added.

3. A biotin-labeled enzyme, such as alkaline phosphatase or peroxidase, is added.

4. Depending on which biotin-labeled enzyme was used in the previous step, either a chromogenic or chemiluminescent substrate is added, and either the color change or the light produced as a consequences of the conversion of substrate into product is measured.

Detection of Mycoplasma

• The nucleic acid-based techniques have several advantages over cultured-based methods, including rapid results, low detection limits and specific organism detection.• This is critical in a hospital setting, since rapid pathogen detection is

important for faster and improved patient treatment and consequently for shortening hospitalization time.• In particular, for PCR-based detection test, selection of the appropriate

target sequences for amplification appears to be of major concern.

Detection of Mycoplasma (Cont’d)

Diagnosis of Malaria

• Currently, malarial infections are diagnosed by either microscopic examination of blood smears or immunological detection of parasite antigens, effective but labor-intensive and time-consuming processes.• Although immunological procedures for

Plasmodium detection, such as ELISAs, are rapid and amenable to automation, they do not always discriminate between current and past infections.• The most intensely labeled hybridizing colonies

were selected because they were expected to contain repetitive DNA.

Detection of T. cruzi

• Diagnosis of acute Chagas disease is usually made by microscopic examination of a fresh blood sample.• Alternatively, a test that takes a longer time but ensures that the

parasite has not been overlooked entails feeding a patient’s blood to uninfected insects’ intestines for parasites about 30 to 40 days later.• In one of the PCR-based assay procedure, a 188-base-pair (bp) DNA

fragment that is present in multiple copies in the T.cruzi genome but is absent from the genomic DNA of several related parasites is the target sequence.

Portable, Rapid Zika Test!! Immunoassays are often not specific

to Zika, reacting to other flaviviruses. The gold standard test, which uses

RT-PCR, typically requires access to expensive lab equipment.

The test, which involves amplification of viral RNA in a chemically heated cup and detection via a color-changing dye, was capable of detecting virus in saliva sample at a sensitivity of 5 plaque-forming units (PFU) in under 40 minutes.

Removal of Nucleic acid

• Regulatory authorities generally insist that the nucleic acid content present in the final preparation, at most, a few picograms per therapeutic dose.• Two types of removal technics: Precipitation (PEI) Treatment with nucleases

DNase IDNase I is an endonuclease that digests single- and double-stranded DNA. It hydrolyzes phosphodiester bonds producing mono- and oligodeoxyribonucleotides with 5’-phosphate and 3’-OH groups.• The enzyme activity is strictly dependent on Ca2+ and is activated by

Mg2+ or Mn2+ ions:in the presence of Mg2+, DNase I cleaves each strand of dsDNA

independently, in a statistically random fashion;in the presence of Mn2+, the enzyme cleaves both DNA strands at

approximately the same site, producing DNA fragments with blunt ends or with one or two nucleotide overhangs.

Benzonase® EndonucleaseAttacks and degrades all forms of DNA and RNA.Is free of detectable proteolytic activity.Is effective over a wide range of operating conditions.Possesses an exceptionally high specific activity.Is manufactured under strictly regulated conditions in order to meet industrial

requirements for a reliable supply and consistent high quality.Is ideal for a variety of applications including:Purification of proteins and other biologicals.Reduction of viscosity caused by nucleic acids.Sample preparation in electrophoresis and chromatography.