Molecular Tests PDF

CLINICAL DIAGNOSIS PATHOLOGY | DR. LINDA D. TAMESIS 1 MOLECULAR TESTS If it is small, it can be missed. Techniques that analyze biological markers in the...

CLINICAL DIAGNOSIS PATHOLOGY | DR. LINDA D. TAMESIS 1 MOLECULAR TESTS If it is small, it can be missed. Techniques that analyze biological markers in the Used to diagnose Down and Turner Syndrome genome and proteome. Observed Characteristics: Molecular techniques ○ Absolute sizes of chromosomes ○ Karyotyping ○ Position of centromeres ○ Fluorescence in Situ Hybridization ○ Relative size of chromosomes ○ Polymerase chain reaction (PCR) ○ Basic number of chromosomes ○ Macromolecule Blotting and Probing ○ Number and position of satellites ○ Arrays ○ Degree and distribution of GC content (important ○ Expression Cloning component of nucleic acids) ○ Next Generation Sequencing Note: Gel Electrophoresis is not a molecular technique BANDING TYPES ○ A technique used to, in addition to molecular Di erent techniques used to visualize and do techniques in order to identify things karyotyping properly G-banding Dark, heterochromatic KARYOTYPE Late 2 di erent definitions: A-T rich ○ Normal genetics of a person: an individual's Most common stain that we use is giemsa complete set of chromosomes. stain ○ Test: Used to describe a laboratory procedure in With giemsa, all of the adenosine and which a laboratory-produced image of a person's thymine will stain dark chromosomes is isolated from an individual cell and R-banding Light (because of giemsa), euchromatic arranged in numerical order. Early Somatic number (N) in humans = 23 G-C rich (very light in color) Euploidy (true numbers) in humans = 46 G-banding and R-banding are Aneuploidy hand-in-hand ○ Either additional chromosome or missing Cannot do G-binding without R-binding chromosome ○ Problem in numbers C-banding Stained with Giemsa ○ Ex: Down Syndrome C-bands are put through alkaline Deletions and Translocations denaturation and depurinization which ○ Not a change in numbers or “ploidy” will let us see the centromere ○ Changes the appearance (not numbers) and Not commonly used function of the chromosomes Q-banding Uses a di erent stain - Quinacrine stain “Ploidy”= numbers SAMPLE CYTOGENETICS Heparinized peripheral blood The study of chromosomes (number) and their structure. Bone marrow aspirates Cytogenetic Testing Tissue biopsies (skin, kidney, liver, muscle and lung) ○ Involves the analysis of cells in a sample of fluid Amniotic fluid (cells or tissues into the fluid) to identify any Chorionic villus sample from developing placenta changes in an individual’s chromosomes. Fetal blood from the umbilical cord Two Important Techniques: ○ Routine karyotyping KARYOTYPING PROCEDURE ○ Fluorescent in situ hybridization (FISH) Other Cytogenetic tests: ○ Comparative genomic hybridization (CGH) ○ Array comparative genomic hybridization (aCGH) KARYOTYPING Basic cytogenetic test One of the first technique to look at somebody’s chromosome This method uses light microscopy and standardized staining procedures on cells in the metaphase (why metaphase?), when the chromosomes are lined along the equator of the nucleus prior to separation and are most 1. Culture cells in nutrient-enriched media to promote condensed (easy to distinguish with one another) cell division in vitro. Identifies changes in number and large 2. Isolate the chromosomes from the nucleus of the translocations cells, place them on a slide, and treat with a special ○ Only large translocations can be detected. stain (Giemsa or Quinacrine stain). ALL PASSED CUTIE! © MED TRANS  CLINICAL DIAGNOSIS PATHOLOGY | DR. LINDA D. TAMESIS 2 3. Take microphotographs of the chromosomes. NORMAL MALE 4. In jigsaw puzzle fashion, rearrange the pictures of KARYOTYPING the chromosomes to match up pairs and arrange by using scissors, them by size, from largest to smallest, followed by they cut out all of the sex chromosomes. these, and match them by where the CLINICAL CYTOGENETICS centromere is Cytogenetic abnormalities may be found in: EXTREMELY Normal individuals DIFFICULT! Patients with phenotypic anomalies Patients with diagnosed genetic disorders NORMAL FEMALE KARYOTYPING Testing may be done during: Prenatal period Postnatal period Childhood and adulthood PRENATAL CYTOGENETICS Comments: Screening for maternal As a female gets older, she is TURNER SYNDROME age-related risk more prone to have children (45, XO in females) with down syndrome Family history of or previous - Aneuploidy child with chromosomal (change in number, abnormality there is an extra X or Abnormal levels of AFP Tumor marker but is also a Y); There are 3 sex (alpha-fetoprotein) in a normal marker if you have a chromosome screening test fetus. If the fetus has a very high level of AFP, it can signify a neural tube defect. DOWN SYNDROME A fetal abnormality detected - (Trisomy 21) on ultrasound A parent who is a carrier of Angelman syndrome unbalanced gametes Prader willi syndrome A parent who is a carrier of - X-linked genetic disorder POSTNATAL CYTOGENETICS Presence of multiple congenital anomalies Suspected aneuploidy (e.g., features of Down syndrome) Doing Karyotyping is quite easy in terms of identification of ADULT AND CHILDHOOD CYTOGENETICS chromosomal abnormality in numbers. More tedious if one is looking for deletions or Comments: translocations Unexplained developmental - delay RESIN STAINS Suspected unbalanced Prader-Willi syndrome Karyotyping but with the use of resin stains. autosome ○ We can use it with fluorescence stain, illuminate it Suspected sex chromosomal Turner syndrome and connect all together abnormality ○ This makes it easier because with the stain we now Suspected fragile – X - see which is higher in content and matching is syndrome easier. Anticipation- only four of these diseases has triple nucleotide However sometimes, even with resin stains identification repeat; it's getting worse in every generation of a problem (such as deletion or translocations) are still quite a problem. Thus, this technique is still better for Infertility Sex chromosomal abnormality chromosome number problems than deletions/ Multiple spontaneous Parents as carriers of balanced translocations. abortions translocations ALL PASSED CUTIE! © MED TRANS  CLINICAL DIAGNOSIS PATHOLOGY | DR. LINDA D. TAMESIS 3 FLUORESCENCE IN SITU HYBRIDIZATION (FISH) Numerical aberrations or translocations in chromosomes Guide cancer treatment Detection of infectious diseases ○ Far superior than usual karyotyping FISH APPLICATION EXAMPLES DNA probe labeled with fluorescent dye and then incorporated into the DNA as long as it has its template there. This can be seen using the microscope. A combination of molecular and cytogenetic technologies that has expanded the ability to investigate chromosome anomalies. PHILADELPHIA Chromosome 9 and 22 ○ Able to detect things that we have trouble CHROMOSOME ○ ABL detecting using karyotyping ○ BCR Uses a probe - a labeled fragment of nucleic acid large Chromosome clumped together enough to hybridize or bind to only those parts of the meaning there was a translocation chromosome with high degree of sequence similarity (Yellow spot/light) Used to detect and localize the presence or absence of specific DNA sequences on chromosomes ○ The most common goal is to determine whether a gene, a specific mutation, or a particular chromosomal rearrangement is present, so the molecular probes used must be well characterized and specific to the locus in question. FISH METHODOLOGY TRISOMY 21 Red - 21 Green -13 (another genetic disease) There are three 21 (red), supposedly there should only be 2. In this technique identification is easier. ○ FISH probes produce a fluorescent dot on the chromosome to which they hybridize. Thus, every pair of chromosomes (or chromosome regions) produces two dots. ○ These double dots sometimes fuse to form one signal. Cells that are monosomic for the 1. Fixation chromosomal region in question 2. Embedding would show only a single dot per 3. Sectioning nucleus, while trisomic cells 4. Hybridization would show three dots. 5. Washing ○ FISH is useful also for number or 6. Microscopy “ploidy” problem and also for even minor or smaller USES OF FISH translocation or deletions cases Form a diagnosis, evaluate prognosis Evaluate remission of cancer (especially breast cancer) ALL PASSED CUTIE! © MED TRANS  CLINICAL DIAGNOSIS PATHOLOGY | DR. LINDA D. TAMESIS 4 NUCLEIC ACID AMPLIFICATION TECHNIQUES temperature at which the PCR thermostable DNA polymerase ○ Best known initiates chain elongation. Target Amplification Typical elongation temperature is Probe Amplification approximately 70 to 72°C. The Signal Amplification polymerase adds free nucleotides to the 3' ends of the primers, using the POLYMERASE CHAIN REACTION (PCR) TERMINOLOGIES DNA template to determine which Components: base to add next. Complementary ○ Templates - DNA bases to the template sequence (A ○ Primers - short DNA fragments with a defined with T, C with G) are added to the sequence complementary to the template or target elongating PCR product. DNA that is to be detected and amplified (which will help us to bind to our DNA sample) NUCLEIC ACID AMPLIFICATION ○ Nucleotides - building blocks Target Amplification ○ DNA polymerase - (for Conventional PCR) links ○ single enzyme or multiple enzymes synthesize individual nucleotides together to form the PCR copies of target nucleic acid (what we basically use product. for COVID, TB, most of viruses) ○ Bu er Probe Amplification ○ amplification products contain a sequence only PRC PROCESS present in the initial probes Signal Amplification ○ concentration of the probe or target does not increase, only the labeled molecules attached to target nucleic acid increases VISUALIZATION (How do we see Nucleic Acid Amplification?) (1) staining of the amplified DNA product with a chemical dye such as ethidium bromide, which intercalates between the two strands of the duplex (2) labeling the PCR primers or nucleotides with fluorescent dyes (fluorophores) prior to PCR amplification. ○ There are 2 ways to stain it. The product or the target 1. DENATURING Extremely hot (95°C) The mixture of starting material (typically DNA from a patient sample) is heated to 95°C to denature the double-stranded template DNA into single strands. Double stranded DNA, you break it down to two which 2. ANNEALING Relatively lower temperature happens in the denaturing stage. Then you’re going to (55°C) Enzyme is going to start to work add the primer and there’s going to be annealing stage and rebuild, you’re going to get the where it’s going to start to stick together and then finally building blocks and stick it on to the extending stage where it gets warm. your templates The mixture is cooled to a temperature just below the predicted melting temperatures of the primer pairs, resulting in primer binding to the single-stranded DNA template strands. The thermostable DNA polymerase can bind to the 3’ end of the primers. 3. EXTENSION Medium temperature (72°C) The temperature is raised to a ALL PASSED CUTIE! © MED TRANS  CLINICAL DIAGNOSIS PATHOLOGY | DR. LINDA D. TAMESIS 5 You do it in cycles so this is one cycle. Sometimes we do Very wide use. it in 20, sometimes in 40. That is why with COVID there Detection of mutations and Identifying a disease’s genetic are a lot of complaints that we are overcycling it. modification of DNA tendency That is why we have a lot of false positives with COVID. Iimaging, diagnosis, and Pathogen presence test Overcycling leads to sticking together of the alleles treatment of viral infections which would have been avoided if we have the routine number for Covid testing Recognition of emerging Microbe detection and Important research question for future researchers: How infectious diseases characterization many cycles is the right amount for what you’re trying to Forensic practices Detecting cancer and the locate? degree of the disease Detecting genetic disorders Cloning cDNA or DNA and PCR qPCR RT PCR associated with hereditary nucleotide sequencing (conventional) Real time diseases Quantitative No Yes Template dsDNA dsDNA SsRNA BLOTS AND PROBES Detection Gel Fluorescence “Blotting,” in relation to molecular diagnostics, is a term method electrophoresis that refers to the process of detecting the presence and quantity of DNA, RNA, or protein in cells. End result End point During phase PCR follows Not really a molecular diagnosis but more of an DNA bands Graph of ancillary procedure that works with molecular amplicon diagnosis. Reaction time 3-4 hours 1 hour It refers to the process of detecting the presence and the quantity of DNA, RNA or protein in cells. Enzyme DNA polymerase DNA polymerase Reverse transcriptase DNA Southern Blot Northern Blot Western Blot polymerase Target DNA RNA Protein Cross Yes, open system No, closed system molecule contamination (a lot of (less Sample DNA extraction RNA isolation Protein contamination) contamination) preparation enzymatic extraction Conventional PCR starts with DNA but we now have digestion another type of PCR called RT-PCR. Separation Electrophoresis Electrophoresis Electrophoresis RT-PCR may stand for ‘real time’. Others say it stands Membrane Nylon Nylon Nitrocellulose for reverse transcriptase PCR. So it could be for those material or PVDF two, but if we do it as a reverse transcriptase then you will see and understand the di erence between these Probe label Radiolabel, Radiolabel, Enzyme two. Enzyme enzyme Cross contamination is very common in conventional. Detection X-ray film, X-ray film, Film, cooled This is what we were using in COVID. methods chemi- chemi- CCD, camera, Real time PCR is mostly used for your viruses and luminescence luminescence LED, or tuberculosis. (e.g. Gene Expert - RT-PCR for TB) infrared imaging APPLICATIONS FOR PCR system There is a sample of DNA, RNA, or proteins and then do the electrophoresis (not really a molecular technique, a technique used in molecular diagnosis). And then it is transferred to a piece of paper and then look at the dots. ALL PASSED CUTIE! © MED TRANS  CLINICAL DIAGNOSIS PATHOLOGY | DR. LINDA D. TAMESIS 6 MICROARRAYS 1. Thousands of DNA or RNA probes are immobilized in a grid. Array means a collection of things. 2. Probes that specifically bind to targets within these genes of interest are printed in small spots onto solid surfaces. 3. DNA is amplified and labeled, then the labeled product is passed over the printed probe spots. 4. If the probes and the amplified target match, they will adhere to one another in a process known as hybridization. If the labeled product and the probe spots stick together and hybridize, then you will be able to detect hybridization by a change in color. Healthy cell (left) and pathological cell (right) and then get a culture to see those which are not present in the cell (gray), present in the normal cell (green), present on both cells (yellow), and in pathological cells only (red). Allows you to do multiple testing or screening to see problems in your DNA and RNA. APPLICATIONS 1. Gene expression profiling 2. Discovery of drugs 3. Diagnostics and genetic engineering 4. Proteomics 5. Functional genomics 6. DNA sequencing 7. Toxicologic research ALL PASSED CUTIE! © MED TRANS