Diagnostic DNA Tests 2024 PDF

Diagnostic DNA Tests Dr. Alawi Habara Assistant Professor College of Medicine Department of Biochemistry IAU 1 Outlines Detecting human Genetic diseases Biochemical tests Genetic tests Amniocentesis Chronic villus sampling (CVS) Routine techniques...

Diagnostic DNA Tests Dr. Alawi Habara Assistant Professor College of Medicine Department of Biochemistry IAU 1 Outlines Detecting human Genetic diseases Biochemical tests Genetic tests Amniocentesis Chronic villus sampling (CVS) Routine techniques Allele-specific cleavage Allele-specific oligonucleotide hybridization Allele-specific PCR Quantitative PCR (qPCR) SYBR Green TaqMan DNA microarray Diagnosis of infectious diseases COVID-19 TB 2 Detecting Human Genetic diseases Biochemical tests Some Genetic disorders can be detected by biochemical tests for the presence of excess substrate or the lack of product Example: Screen for Phenylketonuria (PKU). The defect is in the enzyme phenylalanine hydroxylase c.1222C>T (p.Arg408Trp) The screen for PKU in newborns is done on the 3rd day and is done by direct measurement of phenylalanine in blood H Phenylalanine hydroxylase H – – CH 2 C COO CH 2 C COO NH3+ NH3+ HO Phenylalanine Tyrosine 3 PKU Example of a biochemical test to detect genetic diseases It is a rare autosomal recessive disorder In the newborn, the consumed proteins are broken down into different amino acids, including phenylalanine In days, phenylalanine accumulation causes brain damage Early detection for PKU can prevent brain damage by putting the baby on a therapeutic A screen for PKU in newborn diet that has low phenylalanine 4 Obtained from: Life: The science of biology. 12th ed A mutation in some diseases is located in c.1000A>C. What is the likely position of the amino acid that can be affected by such mutation? ⓘ Start presenting to display the poll results on this slide. 5 Detecting Human Genetic diseases Genetic tests Genetic screening can also be done to detect gene mutation directly at the DNA level The advantage of such testing Any cell can be tested at any time of the life cycle DNA tests allow early detection; prenatal diagnosis 6 Amniocentesis & Chronic Villus sampling Both techniques are used to obtain samples for prenatal diagnosis (mostly used to determine genetic abnormalities) Both are morbid procedures that should be preceded by appropriate counseling. Amniocentesis is a diagnostic technique Chronic villus sampling (CVS) is a procedure in which for taking amniotic fluid from the small samples of the placenta are obtained for uterine cavity using a needle. prenatal genetic diagnosis. Images obtained from https://www.mayoclinic.org 7 Images obtained from https://www.uptodate.com/contents/chorionic-villus-sampling Information are obtained from UpToDate. Amniocentesis & Chronic Villus sampling Chronic villus sampling Amniocentesis Counselling Both procedures are associated with morbidity, and appropriate counseling is a must. Time As early as 10 weeks As early as 15 weeks, some references say week 13 Risk of fetal loss TC-CVS has more risk of fetal loss Less risk when compared to TC-CVS Limb-reduction defect The risk with TA-CVS seems to be similar Blood group If the mother is Rh negative, the mother should receive anti-D Rh antibodies. 8 Information are obtained from UpToDate. RhD and pregnancy 9 Obtained from: https://illustrated-glossary.nejm.org/term/red- cell alloimmunization Detecting Human Genetic diseases Newborns can be screened for genetic mutation The parents can be screened for the heterozygosity for the genetic mutation Routine methods for DNA tests: Allele-specific cleavage Allele-specific oligonucleotide hybridization Allele-specific PCR These methods can only detect a known mutation. 10 Chromosomes This also means that you inherited one allele from your mom and one allele from your dad 11 Allele-Specific Cleavage It is a PCR-based technique Healthy individual βA/ βA MstII The base changes that lead to gene mutation may also 5` 3` cause an alteration in the restriction site C C T G A G G G G A C T C C 5` This will lead to the generation of DNA fragments that are 3` different in length Steps in using this method C C T G A G G 1st PCR is performed to flank the area of your target gene G G A C T C C Restrictions enzymes are used on the amplified PCR DNA product Gel electrophoresis is used to visualize the fragments Sickle cell anemia βs/ βs MstII 5` 3` C C T G T G G Not very G G A C A C C 3` 5` specific. WHY? Can not be cut due to the nucleotide change 12 Allele-Specific Cleavage Paternal Allele Maternal Allele Healthy individual βA/ βA MstII MstII 5` C C T G A G G 3` 5` C C T G A G G 3` G G A C T C C 5` G G A C T C C 5` 3` 3` C C T G A G G C C T G A G G G G A C T C C G G A C T C C 13 Allele-Specific Cleavage Paternal Allele Maternal Allele Sickle cell anemia βs/ βs MstII MstII 5` C C T G T G G 3` 5` C C T G T G G 3` G G A C A C C 5` G G A C A C C 5` 3` 3` Can not be cut due to Can not be cut due to the nucleotide change the nucleotide change 14 Allele-Specific Cleavage Paternal Allele Maternal Allele Sickle cell trait βA/ βs MstII 5` 3` MstII C C T G A G G G G A C T C C 5` C C T G T G G 3` 3` 5` G G A C A C C 5` 3` C C T G A G G Can not be cut due to C C the nucleotide change G G A C T 15 Allele-Specific Oligonucleotide Hybridization Oligonucleotides are made to be complimentary to the denatured DNA to either normal or mutated gene For allele-specific hybridization, usually, these oligonucleotides are fluorescently labeled It’s easier and faster than allele-specific cleavage Can detect a known sequence change Spots contain DNA samples from patients C C T G A G G A G C C T G T G G A G Normal Sickle cell Probe Probe Hybridization and wash Hybridization and wash C C T G A G G A G C C T G T G G A G G G A C T C C T C G G A C A C C T C 16 Obtained and modified from: Life: The science of biology. 12th ed Allele-Specific Oligonucleotide Hybridization Is allele-specific oligonucleotide This means there was hybridization hybridization more or less specific than allele-specific cleavage? Give a brief explanation. This means there was no hybridization Mother Father Child Fetus A normal Probe was used A sickle cell Probe was used Based on this, what each member’s condition will be? 17 Which of the following methods can detect sickle cell anemia with high accuracy? ⓘ Start presenting to display the poll results on this slide. 18 Allele-Specific PCR Basic principle DNA polymerase relies on the last base of the primer matching to the template for it to start building new DNA strands. A Mismatch in the last base means the PCR reaction will not occur. PCR will only occur in the test tube where the perfect matching primer is present. Gel Electrophoresis Perfect match T Mismatch T A G T C No product in gel electrophoresis Give product in gel electrophoresis No exponential amplification Amplification occurs (exponential) 19 Can you detect heterozygous allele using one pair of primers in allele specific PCR? ⓘ Start presenting to display the poll results on this slide. 20 Allele-Specific PCR Homozygous for one allele (Allele A) Paternal Allele Maternal Allele T T A A T T C C 21 Allele-Specific PCR Homozygous for one allele (Allele C) Paternal Allele Maternal Allele T T C C G G C C 22 Allele-Specific PCR Heterozygous individual Paternal Allele Maternal Allele T T C A G T C C 23 Allele-Specific PCR 2 pair of primers T A T C T C G C T A T C T C G 24 C Quantitative PCR Quantitative PCR (qPCR) is a method to measure the quantity of DNA in a sample by amplifying a target DNA sequence. It uses the PCR principle to amplify a specific DNA target Then, it detects the DNA amplification in real time using fluorescent dyes or fluorescent probes that bind to the DNA There are mainly two types of fluorescent detections for qPCR Intercalating dyes (SYBR green) Sequence-specific probes (TagMan) Quantitative PCR (qPCR) can also be referred to as Real Time PCR (RT-PCR) Quantitative Real-Time PCR (qRT-PCR) Do not confuse this with Reverse Transcriptase qPCR (RT-qPCR) 25 Quantitative PCR SYBR green-Principle Step 1 Denaturing 95 ºC Lower Ct value = Higher amount of DNA Step 2 Annealing 50 ºC Step 3 Extending 72 ºC key differences with SYBR Green qPCR The presence of SYBR green dye in the reaction mix. SYBR green will bind to the double-stranded DNA The machine will detect the amount of SYBR green florescent after each cycle 26 Quantitative PCR TaqMan Principle Lower Ct value = Higher amount of DNA Step 1 Denaturing 95 ºC Step 2 Annealing 50 ºC Step 3 Extending 72 ºC key differences with TaqMan qPCR The presence of a Fluorescent labeled Probe that is specific to the target gene, SNP, or mutation The fluorescent attached to the probe will be released as TaqMan polymerase starts building the new DNA strand because it has 5` to 3` exonuclease activity The amount of Fluorescent released corresponds to the number of targets being amplified. The machine will detect the amount of florescent released from the 27 probe after each cycle Quantitative PCR SYBR Green VS TaqMan Principle 28 Obtained from: https://www.youtube.com/watch?v=n14TYAO5N3g What are the differences between SYBR green and Taqman qPCR? ⓘ Start presenting to display the poll results on this slide. 29 DNA fingerprinting Individuals can be definitively characterized by their DNA sequence; this is called DNA fingerprinting. Whole genome sequencing can not be done as a routine method for DNA fingerprinting. However, sequences that are highly variable (polymorphic) can be used as DNA fingerprinting as they will differ from one individual to another. A PCR method is used to amplify the repeated sequence. These two highly variable (polymorphic) sequences can be of two types: Single nucleotide polymorphisms (SNPs) Short tandem repeats (STR) STR are also known as microsatellites. They are simple DNA fingerprinting can be used for: sequence that is repeated, and they involve 1-6bp. Criminal investigation/Forensic medicine Generally, they are found in non-coding regions Paternity 30 A crime has been committed, and in the crime scene, the DNA of the criminal was found. Test for STR was done to figure out the criminal out of the 3 suspects. Which one do you think has committed the crime? ⓘ Start presenting to display the poll results on this slide. 31 DNA fingerprinting 32 STR can be also used in paternity tests. If the father of a child is not known. Which one do you think is the father of the child? ⓘ Start presenting to display the poll results on this slide. 33 DNA fingerprinting  ‫ﻻ ﻣو واﺿﺢ ﻣن ﺷﻛﻠﮫ‬ Paternity test !‫!! ﻻزم ﺗﺳوي اﻟﻔﺣص‬ 34 Can you identify identical twins using genetic/molecular tests? ⓘ Start presenting to display the poll results on this slide. 35 DNA microarray Basically, it is an automated version of the hybridization technology. It can be used to detect both DNA and RNA (but is most commonly used for RNA expression these days). Hybridization and washing 36 & Metabolism. Obtained from: Vlacich G et al, Nature Clinical Practice Endocrinology 2007 Aug DNA microarray Healthy cell Pathological cell Cell types Culture RNA isolation Reverse transcriptase with fluorescent tagging (cDNA synthesis) 37 Image by sagar Aryal, created using biorender.com DNA microarray Applications Gene expression profiling It allows checking multiple RNA transcripts all at once. RNA from the cells is converted to cDNA that is tagged with fluorescent and then hybridized into a microarray chip. DNA variation screening Assay known mutations to specific disease Check multiple human SNPs Microarray Sample Objectives Applications Type Expression mRNA Determine the gene expression change between Cancer diagnostic and prognosis profiling disease and health condition Response to treatment SNP analysis Genomic Detect polymorphism Determine genetic predisposition to DNA diseases Array CGH Genomic Identify genetic duplication, deletion, and copy Unbalanced genetic abnormalities (gain or DNA number change loss of genetic material) 38 Diagnosing of infectious diseases Tests based on DNA probes and PCR technology Used to show whether the DNA of an infectious agent is in blood or a tissue sample. Only a small target sequence (template) from the pathogen is required, and specific primers (which much match the pathogen DNA) This means the test is extremely sensitive 39 COVID-19 detection During the COVID-19 pandemic, molecular-based detection for SARS- Cov2 RNA was extremely important in controlling the spread of the pandemic. The RT-PCR method was the 1st to be utilized. Then, faster methods were used, isothermal nucleic acid amplification testing (iNAAT) and NGS. PCR-based methods are highly sensitive. 40 Palak Shah et al. Circulation. 2020 © 2020 American Heart Association, Inc. COVID-19 detection 41 Obtained from: https://www.youtube.com/watch?v=xiTQ6-MOgdk What do you think is the advantage of molecular-based detection for infections such as COVID-19 during the pandemic over antigen-based or antibody-based testing? Explain your answer. ⓘ Start presenting to display the poll results on this slide. 42 TB detection Advances in molecular biology have enabled rapid and accurate detection of Mycobacterium tuberculosis complex (MTBC) and nontuberculous mycobacteria (NTM), as well as their drug resistance. Molecular tests have improved TB diagnosis, particularly in cases with negative microscopy results. Despite advancements, the WHO advises that molecular tests are not replacements for traditional microbiological cultures and phenotypic antimicrobial susceptibility testing. WHO-Recommended Rapid Diagnostic Tests: These tests detect M. tuberculosis and resistance to rifampicin (RIF) and fluoroquinolones. Used to diagnose TB, guide treatment, and monitor treatment response. Nucleic-Acid Amplification Tests (NAATs): Most WHO-endorsed rapid TB tests use NAAT technology 43 Obtained from: Bartolomeu-Gonçalves G et al, Diseases. 2024 Sep References Hillis DM, Heller CH, Hacker SD, Hall DW, Laskowski M, Sadava DE. Life: The science of biology. 12th ed. Sunderland, MA: Sinauer Associates; New York: Macmillan Learning, 2020. Ghidini A. Chronic Villus sampling. UpToDate: Waltham, MA; 2023 [cited 2023 -9-31]. Available from: https://www.uptodate.com/contents/chorionic-villus-sampling Ghidini A. Diagnostic aminocentesis. UpToDate: Waltham, MA; 2023 [cited 2023 -9-31]. Available from: https://www.uptodate.com/contents/diagnostic-amniocentesis Arishi WA, Alhadrami HA, Zourob M. Techniques for the detection of sickle cell disease: a review. Micromachines. 2021 May 5;12(5):519. Fan H, Chu JY. A brief review of short tandem repeat mutation. Genomics, proteomics & bioinformatics. 2007 Jan 1;5(1):7-14. Vlacich G, Roe C, Webb GC. Technology insight: Microarrays—Research and clinical applications. Nature Clinical Practice Endocrinology & Metabolism. 2007 Aug;3(8):594-605. Shah P, deFilippi CR, Mullins KE. Navigating COVID-19 testing: special considerations for the cardiovascular clinician. Circulation. 2020 Dec 15;142(24):2293-5. Bartolomeu-Gonçalves G, Souza JM, Fernandes BT, Spoladori LF, Correia GF, Castro IM, Borges PH, Silva- Rodrigues G, Tavares ER, Yamauchi LM, Pelisson M. Tuberculosis Diagnosis: Current, Ongoing, and Future Approaches. Diseases. 2024 Sep 3;12(9):202. 44 Thank you, Any questions? Contact [email protected] Office: 031-33-31126 Office location: Building A72 college of medicine, Biochemistry department office #3006 45