Genetic Diagnosis & Lab Techniques

Diagnosis of Genetic Diseases

• Diagnosing a genetic condition involves assessing physical characteristics, family history, and screening test results.
• A comprehensive clinical examination is necessary for diagnosis.

Physical Examination in Genetic Diagnosis

• Specific physical traits like distinctive facial features may suggest a genetic disorder.
• Taking measurements such as head circumference, distance between the eyes, and limb length may be required.
• Neurological and eye exams, as well as imaging studies like X-rays, CT scans, and MRI can aid diagnosis.

Equipment Used in Genetic Disease Diagnosis

• PCR (Polymerase Chain Reaction)
• RT-PCR (Reverse Transcription Polymerase Chain Reaction)
• Nanodrop Spectrophotometer
• Gel electrophoresis
• Gel documentation system
• Centrifuge
• Spin
• Water distillation apparatus
• Vortex mixer
• Sensitive electronic balance
• Microwave

Polymerase Chain Reaction (PCR)

• PCR is a technique used to create millions of copies of a specific DNA segment.
• Developed in 1983 by Kary Mullis, who won the 1993 Nobel Prize in Chemistry.
• It was used during the COVID-19 pandemic to detect viral infections.

Reverse Transcriptase PCR (RT-PCR)

• RT-PCR is an amplification method that transcribes RNA into complementary DNA (cDNA), which is then amplified using PCR.

PCR vs. RT-PCR

• PCR amplifies a DNA segment to create millions of copies of a DNA sequence, while RT-PCR detects gene expression in molecular biology.
• PCR consists of denaturation, annealing, and extension steps, and is followed by PCR in RT-PCR.
• PCR uses a double-stranded DNA molecule as template, whereas RT-PCR uses a single-stranded RNA molecule for reverse transcription.
• DNA Polymerase is the enzyme used in PCR, and reverse transcriptase and DNA polymerase are enzymes used in RT-PCR.
• PCR aids functional analysis of genes, diagnosis, monitoring of heredity diseases, DNA cloning, and DNA sequencing; RT-PCR is used in detection of gene expression.

NanoDrop Spectrophotometer

• A common lab instrument that measures DNA, RNA, and protein concentration using 1–2 µL samples.
• It measures light absorption at 260 nm to determine the amount of DNA in a sample.
• A ratio of ~1.8 is considered "pure" for DNA, while ~2.0 is considered "pure" for RNA; lower ratios may suggest contamination.

Gel Electrophoresis

• A technique used to separate DNA, RNA, or proteins based on molecular size.
• The two primary types are Polyacrylamide Gel Electrophoresis (PAGE) and Agarose Gel Electrophoresis (AGE).
• For agarose gel electrophoresis, a 0.8% gel provides good separation for 5-10kb DNA fragments, while a 2% gel offers good resolution for small (0.2-1kb) fragments.
• A 1% gel is often used for standard electrophoresis.

Gel Documentation

• A method to record and measure labeled nucleic acids and proteins in media like acrylamide and agarose.
• DNA or RNA is typically stained with ethidium bromide, visualized under UV light.

Spin Mini Centrifuge

• A small, powerful centrifuge used to isolate sample components.
• Compatible with 2.0ml, 1.5ml, 0.5ml, and 0.2ml centrifuge tubes within an 8-place tube rotor.

Centrifuge

• A laboratory instrument driven by a motor, used to spin liquid samples at high speed.
• It works on the sedimentation principle, separating substances of different densities via centripetal acceleration.

Electronic Balance

• Used to find precise weight measurements, particularly for weighing chemicals in laboratory experiments.

Distillation

• A process to purify water by evaporation.
• It involves heating contaminated water to form steam, leaving behind inorganic compounds and non-volatile organic molecules. The steam is then cooled and condensed into purified water.

Microwave

• Used for rapidly boiling media for approximately 5 seconds.
• Over-microwaving should be avoided.
• The solution should appear clear, without any visible media granules once complete.

Uses of Genetic Testing

• Newborn Screening
• Carrier Testing
• Prenatal Diagnosis
• Diagnostic/Prognostic testing
• Predictive/Predispositional testing

Explore genetic disease diagnosis through clinical exams and PCR. Learn about RT-PCR, NanoDrop spectrophotometry for protein concentration, and gel electrophoresis for molecule separation. Understand the use of centrifuges and electronic balances in labs.