Molecular Techniques

What are the basic steps for DNA isolation?

Lyse cells: -Detergent dissolves cell membrane and denatures protein. -EDTA chelates divalent cations required by nucleases. -Proteinase K degrades proteins. Extraction: -Organic: uses phenol:chloroform-isoamyl alcohol, dissolves hydrophobic contaminants, denatures and removes proteins, aqueous phases: organic phase, DNA precipation -Nonorganic: Protein precipitation and DNA preciptiation

Northern Blot

RNA and it is rarely used

CA-125

-ovarian cancer -monitors responsiveness to chemotherapy

PCR: hybridization for bacteria DNA

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Cancer antigens

Biological substances synthesized and released by cancer cells or substances produced by the host in response to cancerous tissue

What are the storage requirements for DNA?

Blood, bone marrow, and Fluids: - < 1 day, 23 C; 3 days, 4 C -WBC, > or equal to 1 year, -20 C or -70 C. Only WBC are frozen, red cells will lyse when thawed.

Discuss potential problems with isolating DNA

Degree of purity of DNA does it contain contaiminant? Integrity of the DNA- HMW vs. LMW (mechnical shearing and DNase enzymes degrade DNA into smaller pieces) Yield (quantity of DNA prepared from a given amount of starting material)

Molecular Diagnostics (MD)

The use of molecular biology techniques to expand scientific knowledge of the natural history of diseases, identify people who are at risk for acquiring specific diseases, and diagnose infectious and other human diseases at the nucleic level.

Cancer antigen phase one

induction-exposure to carcinogen

What is the preferred anti-coagulant for molecular diagnostic specimen testing?

-Blood: EDTA for virology and molecular biology (lavender) -FISH: sodium heparin for cytogenetic studies and molecular studies (brown) -Bone Marrow: acid citrate dextrose (ACD) solution for molecular biology (yellow)

Ideal tumor markers

-Easy and inexpensive to measure in readily available fluids -Specific to tumor and commonly associated with it -Have proportional relationship between plasma level and tumor mass -Have abnormal level in presence of disease but without clinical presentation (early detection before the patient shows symptoms) -Stable levels that are not subject to fluctuations -In healthy individuals, they exist in much lower concentrations

Southern Blot

-Electrophoretically separated DNA fragments are blotted to nitrocellulose membrane -Hybridized with radiolabeled single-stranded DNA complementary sequences -Double stranded DNA is detected by radiography -Application for diseases with 50-100 base pair changes in DNA -Fragile X, T or B cell lymphomas, sickle cell, Hemophilia A

What is the difference between annealing and hybridization?

-Hybridization is the interaction between two single-stranded nucleic acid molecules to form a duplex (double-stranded) molecule based on the complementary base pairing of their respective sequences. -If neither DNA strand is labeled, the process is called annealing -When one DNA strand is labeled, the labeled strand is referred to as a probe and the process is called hybridization because a hybrid molecule is formed between a labeled and unlabeled strand.

MIcroarray (DNA chip)

-Product of bonding or direct synthesis of specific DNA probes on a stationary silicone support -Mini gene fragments attached to glass chips examine gene activity and ID genetic mutations -Use a reverse-dot hybridization format reaction between the microarray sequences and a fluorescent sample -The chips are scanned with high-speed fluorescent detectors and the intensity of each spot is quantitated -Used for pathogen ID, polymorphism detections, and viral resistance mutation detection

Clinical uses for tumor markers

-Screen for disease in the general population (rarely useful) -Diagnosis in patients with symptoms (biopsies) -Adjunct in clinical staging -indicator of tumor volume -aid for selecting appropriate therapy -monitor response to therapy (common use) -prognostic indicator -early detection of disease recurrence

What are the basic steps for RNA isolation?

-Separate WBCs from RBCs, if necessary -Lyse WBCs or other nucleated cells in presence of protein denaturants, RNase inhibitors -Denature/digest proteins -Separate proteins, DNA, and contaminants from RNA -Precipitate RNA if necessary -Resuspend RNA in final buffer

What are the uses of Branched DNA?

-Target nucleic acid sequences are not replicated through enzymatic amplification. -Detection sensitivity is provided by amplification of the signal from the probe. -Uses "capture probes," "bDNA probes" and "bDNA amplifier probes." -Assay is based upon microtiter plate technology. Isothermal -Signal amplification -A series of hybridizations attaches multiple signals to each target molecule. -HBV, HCV, HIV-1 -Solid-phase sandwich hybridization assay incorporating multiple sets of synthetic probes -Microtiter plates have multiple target specific probes to capture the target (DNA or RNA) -Second set of probes then bind to target -Enzyme labeled probes then hybridize to each branch -Used to measure viral loads of HBV, HVC, HIV-1, CMV, and microbial organisms (T. brucei)

Conditions for Hybrid Formation

-Temperature •Salt concentration •Concentration of nucleic acids •Presence of denaturing agents (formamide) •Presence of HMW polymers (dextran sulfate) increases [nucleic acids] by excluding volume.

Reverse Dot Blot

-Traditionally, amplified sample DNA is bound to a membrane and probed by an ASO probe. Any unbound probe is washed away and a positive test result is observed by a color reaction from an enzyme linked to the probe. -HLA or human papillovirus typing -Biotin is the affinity label which does not generate a signal but interacts with streptavidin or avidin -This complex is conjugated with a signal-generating fluorochrome -Detection system is conjugate-dependent and include colorimetric, fluorescent, or chemiluminescent methodologies

Dot Blot

-amplification analysis -expression analysis (RNA) -mutation analysis

AFP (alpha feto protein)

-associated with hepatocellular carcinoma (HCC), germ cell carcinomas (testicular or embryonal carcinomas, yolk sac tumor, choriocarcinoma) -used as a screening test for liver cancer, cirrhosis, or chronic hepatitis

Describe DNA structure and composition

-consists of a pair of anti-parallel strands (5' to 3' and 3' to 5') -alternating deoxyribose and phosphate groups -covalent linkage by phosphodiester bonds -strands held together by hydrogen bonds -adenine binds thymine (two bonds) -guanine binds cystosine (3 bonds)

PCR: Reverse transcriptase PCR

-convert RNA to DNA for analysis of RNA -useful in detection of RNA viruses

CA 15-3

-correlates with tumor burden -elevated in metastatic breast, pancreatic, lung, colorectal, and liver cancer -monitors responsiveness to chemotherapy

CA 27-29

-discriminates primary breast cancer from healthy subjects better than 15-3. -used with 15-3 for breast cancer

CEA (carcinoembryonic antigen)

-first identified in adenocarcinoma of the digestive tract -Limited use in detecting colon cancer -Elevated in many other non-cancerous conditions -Lacks sensitivity and specificity -used as prognostic indicator and to detect recoccurrence -associated with cancers: breast, bronchus, pancreas, stomach, bladder, prostate, ovarian, neuroblastoma, and thyroid -Methodology EIA and RIA: serial measurement and beware of switching methods

PCR: Nested

-involves the sequential use of two primer sets. The amplicon obtained is then used as the target sequence for a second amplification of a sequence internal to an amplicon. -Allows for extreme sensitivity. And increased specificity -subject to high rate of contamination unless physical separation of the first and second round amplifications are used

CA 19-9

-pancreatic cancer -90% sensitivity and 85% specificity -adenocarcinomas (lung, gastric, biliary, colonic)

Describe RNA Structure

-single stranded -Ribose sugar substituted for deoxyribose of DNA -Uracil replaces thymine of DNA -A:U

PCR: Multiplex

-two or more primer sets are used -used to amplify different targets in the same reaction tube -detection is accomplished using the Southern Blot

Quantitative real-time PCR

-uses fluorescence resonance energy transfer -quantitation of specific DNA sequence -potential to quantify the infectious burden

PSA (Prostate specific antigen)

-very little PSA in normal male -benign prostate hyperplasia has a high Total PSA, but prostate cancer is typically higher -High levels in seminal fluid -bound to serine protease inhibitors -PSA is organ specific not tumor specific, should not be used for cancer screening -used for monitoring progress post-prostatectomy because levels should fall between 1-3 months after surgery

What are the storage requirements for RNA?

Blood, bone marrow, and fluids: - < 2 hours 23 C or 4 C -5 days 23 C; 7 days 4 C in denaturant - 1-2 weeks, -70 C in denaturant (PREFFERED) -WBC, 2-4 weeks, -20 C, > 6 months, -70 C

Potential specimens for testing using molecular diagnostics

DNA and RNA

What information is gathered from spectrophotometer

DNA260/Protein280 ratio tells the purity of the DNA. It wil also allow you to find out the concentration and yield of DNA or RNA

What are the basic steps of PCR?

Denaturation: (90 to 96 C) for 20 seconds. It breaks DNA from its double strand form to its single stranded form. Annealing: (40 to 68 C) for 20 seconds. Allows the hybridization of the primers to their complementary sites on the the single stranded DNA. Vast excess of primers will ensure that the hybridization is between the sample DNA and primers and not between the sample DNA and primers and not between the two strands of test DNA. Extension: ( 70 to 75 C) for 30 seconds. The temperature optimum for Taq polymerase. The polymerase extends the primers in the 5' and 3' direction by incorporating the dNTPs into the growing complementary DNA strands. The process is then repeated 20 to 30 times. Then it can be visualized on anethidium bromide-stained gel.

Cancer antigen phase four

Dissemination-invading cancer spreads to various parts of the body

Discuss potential problems with isolating RNA

Easily degraded by RNAse enzymes. Tissue culture serum contains high levels of RNase (placenta, liver, some tumors). RNase requires 2 hours at 200 C or 30 minutes in 1 M NaOH. Guanidinium isothiocyanate is a denaturing agent that inactivates RNase.

Cancer antigen phase two

In situ- transformed cell develops into cancer, but remains localized and does not invade other tissues (metastasize)

Cancer antigen phase three

Invasion-malignant cells multiply, invade into deep tissues, and gain access to blood vessels and lymphatic channels

Applications of molecular diagnostics

Molecular genetics: -single gene disorders -polygenic disorders -chromosomal disorders Molecular Oncology: -Diagnostic testing -Disease prognosis -Determination of predispostion Hematopathology: -diagnostic testing -determination of clonality Identity testing: -parentage -clinical testing Infectious Disease: -Microbial identity testing -Genotyping/drug resistance testing -qualitative and quantitative detection of infectious agents

What are the uses of DNA sequencing?

Mutations, resistance testing and HLA sequencing "Gold Standard" Method Sanger method most common Displays exact nucleotide or base sequence of a fragment of DNA that is targeted: -Amplification by PCR usually -dsDNA denatured to ssDNA -Add ssDNA primers complementary to target sequence

Spectrophotometer: explain how the quantity of DNA/RNA can be determined

Ratio of DNA/Protein using the absorbance reading at 260/280 nm wavelength

Stringency

includes the conditions of hybridization that control the specificity of binding between two single-strand portions of nucleic acid (usually probe and immobilized target sequence). 1.Increased temperature or decreased ionic strength (salt) increases stringency. 2.Only duplexes with near perfect sequence complementarity can exist at high stringency.

Spectrophotometer: explain how the quality of DNA/RNA can be determined

ratio must be between 1.7 to 2.0 to be consider good quality