Arsalaannaveed Ayesha naeem Bdar slam Sabanaseem Uzairhashmi Application of PCR-technique in biological labs
Applications of PCR in Diagnostics Uzairhashmi
PCR for pathogen detection • other methods for detection • Cultures and colony counting assay • Oldest bacterial detection method • Culturing methods are extensively time consuming • 2. Immunology Based Methods : • Antigen-antibody interactions • Doesn't tell the pathogen load (serum-viral load)
PCR A Nucleic acid amplification technology “Widely used in pathogen detection” Basic steps Isolation of DNA Amplification of DNA Quantification of DNA sequence (targeted pathogen’s genetic material)
Priority over other detection methods • Today the detection methods have been replaced by PCR due to • Quick results (culturing takes even weeks) • More specific (no false positive results, as a result of contamination) • Quantification of serum viral load , (pathogen titter from sample)
PCR pathogen detection assays • Principle :- • Amplifies target nucleic acid sequence from microbes present in the samples • This amplification of target sequence is achieved by • Custom designed probes • Custom designed primers
Contd... First degree of specificity : Achieved by combination of amplification primer sequence Additional degree of specificity : Achieved by the hybridization of the probe To a Region of nucleic acid sequence that identifies the microbe of interest
Specificity and sensitivity Microbe’s genome :- Specificity of microorganism is due to a target (specific) sequence in its genome. This sequence encodes their virulent agents. How to distinguish microbe from its nearest neighbour ? The gene (or its portion) that contribute to a disease phenotype of the pathogen: distinguish a target microbe with its nearest neighbour
Distinction and detection • Target sequence needs to be identified • Highly specific and unique for a particular species (this target sequence will be ) Specificity implies two properties Exclusion Inclusion Assay will detect all strains of target species Assay will not detect neighbour species
Detection of HIV by IN situ PCR HIV has the ability to infect different cell types and tissues Principle : HIV nucleic acid sequence is amplified using specific primer pairs. • Amplification of target DNA • Hybridization with 3’ end labelled oligonuecleotide • Detection with anti-DIG-AP
Amplification of target DNA Amplification of HIV DNA was carried out on slides, using primer pairs from the gag (SK38/39) region (3), and performed on a thermo cycler.
Hybridization Hyb. With DIG-labelled oligonucleotide probe Detection Using polyclonal antibody DIG label was detected by Alkaline phosphate conjugated polyclonal antibody
Detection of HCV RNA in patient specimens by polymerase chain reaction (PCR) provides: • Evidence Of Active HCV Infection • Is Potentially Useful For Confirming The Diagnosis And Monitoring The Antiviral Response To Therapy.
TECHNIQUE USED reverse transcriptase-PCR- (RT-PCR-) based assays Qualitative RT-PCR
Quantification is achieved by: AMPLIFICATION INSIDE THE REACTOR TUBE TWO TEMPLATES IN A SINGLE REACTION TUBE THE INTERNAL STANDARD THE TARGET
Comparison of the final amounts of both templates allows calculation of the initial amount of HCV RNA. • The internal standard is an internal control RNA with nearly the same sequence as the target RNAwith a clearly defined initial concentration. • The internal control is amplified by the same primers as the HCV RNA. • It has a specificity of almost 100%, independent of the HCV genotype
Real-Time PCR Assay for Detection and Quantification of Hepatitis B Virus Genotypes A to G
The detection and quantification of hepatitis B virus (HBV) DNA play an important role in diagnosing and monitoring HBV infection as well as assessing therapeutic response • The great variability among HBV genotypes and the enormous range of clinical HBV DNA levels present challenges for PCR-based amplification techniques
PCR assay designed to provide accurate quantification of DNA from all eight HBV genotypes in patient plasma specimens.
Tuberculosis • Limitation in culturing • Species are slow growing • Needs 6-8 weeks for growing • Species can be contaminated while growing • RESULT :- • Specificity is lost due to contamination • And can also yield false positive results
PCR based TB diagnostic test • Sputum • Sample processing • DNA extraction • AMPLIFICATION of MTB DNA • 123 bp DNA fragment is amplified using specific primers • amplified products were analysed by electrophoresis • 123bp specific band detected by gel doc TRANS illuminator
PCR based dengue detection Identification of dengue virus 2-step PCR reaction 1.Reverse transcription 2.Amplification using universal dengue primers
Reverse transcription RNA strand is reverse transcribed into DNA using reverse transcriptase cDNA is amplified using RT-PCR Universal dengue primers are used Targeting a specific region of viral genome • PCR products are separated on gel electrophoresis • Different size bands are seen • They are compared with a standard marker • Tells the relative molecular mass of nucleic acids • dengue serotypes are identified by the types of bands
RT-PCR is one step assay system Primers and probes Specific for each genotype Detection of pathogens in real time without using the electrophoresis Florescent probes are used
PCR in Prostate Cancer detection Methylation specific PCR (msp) technology DNA is methylated only at certain cytosine located 5' to a guanosine. This occurs especially in GC-rich regions, known as CpG islands Obj :- methylation state of sequence
How to achieve this ? Chemical modification in the cytosine residues in DNA Sod. Bi sulphite will convert all the unmethylated C-sequence residues into uracil Methylated cytosine will remain same Different DNA sequence will be formed for methylated and un-methylated DNA .
PCR primers will distinguish Primers will anneal to the unchanged cytosines (that are methylated in the gene. Primer will anneal with altered cystosine(uracil- that were un-methylated) Comparison will reveal the methylation state of DNA
Primer set with altered sequence gives a product ? Indicated cytosine were un-methylated Primer set with unchanged sequence gives a product? Cytosine was methylatedand protection from alteration
Prostate cancer : aiktak’nikeekharabi Genetic alteration in prostate carcinoma Hyper-methylation of GSTP1 promotor • Function of GSTP1 : • Involved in intracelleular detoxification reaction • Candidate tumor suppressor gene in Pros.Cncr • Hyper methylation results in loss of gene expression GSTP1 : maker for detection and molecular staging of p.c
In Chronic Myelogenous Leukemia Cancer of WBC Increased and uncontrolled growth of myeloid cells in bone marrow. Genetic abnormality Chromosomal translocation , formation of phaliadelpia chromosome BCR gene in 22 , fused with abl gene in 9 p210
p210 Add phosphate group to the tyrosine :tyrosine kinase Activates protien cascade that control cell cycle Inhibits DNA repair .
RT-pcr comes into action • RNA is extracted • Subjected to RT-PCR • 3 –types of primary transcripts of (bcr/abl gene)are amplified • B2a2 • B3a2 • E1a2 If there is no amplification of BCR-abl fusion mRNA result will be reported as negative
RESEARCH APPLICATIONS OF PCR AYESHA NAEEM
GENE CLONING Major application of PCR • PCR can produce large quantities of DNA that can be readily cloned and used to study the functions and behavior of genes in living systems.
THE PCR STEPS Denaturation Annealing (60-70C) Elongation (72C)
PCR-mediated cloning is a family of methods rather than a single technique. • TA cloning • Blunt-end cloning
TA CLONING uses Taq polymerase and Tth DNA polymerase that preferentially add adenine (A) to the 3' ends of the PCR products. These products are cloned into a vector containing complementary overhangs of the base thymidine (T).
BLUNT-END CLONING uses DNA polymerases that possess proofreading activity, such as Pwo DNA polymerase. They remove mispaired nucleotides from the ends of double-stranded DNA and generate blunt-end PCR products.
SELECTIVE DNA ISOLATION PCR allows isolation of DNA fragments from genomic DNA by selective amplification of a specific region of DNA. Thus, PCR provides high amounts of pure DNA to be used as probes for Southern or Northern hybridization and as primers for DNA cloning.
GENE EXPRESSION STUDIES Reverse transcription quantitative polymerase chain reaction (RT-PCR followed by qPCR) is the gold-standard technique for measuring gene expression. • sensitivity • broad dynamic range • lower-cost of instrumentation and reagents
mRNA quantification qRT-PCR is a highly sensitive technique in which a very low copy number of RNA molecules can be detected • RT-PCR first generates a DNA template from the mRNA by reverse transcription, called cDNA. • cDNA template is used for qPCR where the change in fluorescence of a probe changes as the DNA amplification progresses. • With a carefully constructed standard curve, qPCR can produce an absolute measurement of the number of copies of mRNA, in units of copies per nanolitre of homogenized tissue .