Genetic tests😍

Pedigree analysis, Cytogenetics and Molecular Biology

Pedigree Analysis

 

• Pedigree Rules (2G Rule: Gender and Generation):

NOTE:

• consanguineous → ↑ likelihood of AR → 4 - 8%

ChIP-Seq (Chromatin Immunoprecipitation Sequencing)

• Use:
• Identify binding sites of DNA-binding proteins
• (e.g., transcription factors, chromatin remodelers).
• Also maps:
• Histone modifications
• protein-DNA interactions

• Method: Immunopurified DNA sequenced by NGS.

• Variants:
• Standard ChIP-Seq:
• Immunopurified DNA directly sequenced.
• ChIP-Exo:
• Cross-linked protein-DNA treated with exonucleases
• removes non-intimate DNA
• gives single nucleotide resolution of protein occupancy.

Genetics Tests

Molecular Methods

• Reverse Transcriptase PCR (RT-PCR):
• Converts RNA to DNA.
• Detects RNA.

• Real time PCR (qPCR):
• For quantification of nucleic acids.

• Blotting techniques:
• Used to identify specific substances.
• Southern blot: Identifies specific DNA.
• Northern blot: Identifies specific RNA.
• Western blot: Identifies unknown proteins.
• Southwestern blot: Identifies DNA binding protein

• G banding (Giemsa staining of chromosome):
• Used for Karyotyping.
• To detect copy number variations of DNA.
• Eg: Trisomies

Human Genome Project (HGP)

• Duration: 1990–2003 (13 years)

• ~20,500 genes & 3 Billion bps identified & mapped

• Methods: First-generation sequencing (Sanger, SGS)

• Output: Reference sequence for most chromosomes

• Basis for second-generation sequencing (e.g., array techniques)

• Sanger sequencing:
• Used for DNA sequencing.
• Employs
• Dideoxynucleotide Triphosphate method (DdNTP)
• Termination method.
• ddNTPs lack a hydroxyl group on the 3' carbon of the sugar,
• which prevents further nucleotide addition and further elongation of DNA
• DNA sequencing/Protein sequencing is first done by Sanger

Next generation sequencing

• Used now

• Economical

DNase footprinting

• Study DNA Protein Interaction

• Technique steps
• allow a protein to bind to DNA
• treat complex with DNase
• DNase cleaves DNA at random locations

• Protection principle
• enzyme cannot cleave DNA when bound to the protein
• creates a region of protected DNA
• this region = “footprint”
• reveals the specific sequence where the protein is bound to DNA

• Analysis
• separate DNA fragments by gel electrophoresis
• visualize to determine location of the protein–DNA complex

Steps of the Recombinant DNA technique are:

1. Isolation of the gene of Interest

2. Incorporation of the gene into the vector (plasmid)
• This process utilizes Restriction endonucleases/Ligases

3. Insert vector in E. Coli (host)

4. Expression of the gene in E. Coli for the production of protein

5. Bacterial wall lysis

6. Electrophoresis (SDS -PAGE)

7. Western blotting/Hybridisation

8. Elution of protein.

Flow Cytometry

Purpose

• Assesses individual cells:
• cell size,
• granularity, and
• protein expression (immunophenotyping)

• Commonly used in:
• Hematologic abnormalities:
• Leukemia, PNH, fetal RBCs in maternal blood.
• Immunodeficiencies: e.g.,
• CD4+ count in HIV.

Sample:

• Any fluid (blood, BM aspirate, CSF, pleural fluid).

Principle: 

• Cells are tagged with fluorescent-labeled antibodies specific to surface or intracellular proteins

• Hydrodynamic focusing (cells fall one by one).

Light Source: 

• Blue light (488 nm wavelength)

• Laser excites fluorochromes.

Measurements:

• Forward Scatter (FSC):
• Cell size

• Side Scatter (SSC):
• Cell complexity/granularity

Graphs:

• History (Histogram) Flows (FC) → But has dots () in it

• Histogram:
• Shows peaks
• EMA (eosin-5-maleimide) binding test
• for hereditary spherocytosis
• hEMA → HS

• Dot Plot Analysis:
• Cells as dots;
• quadrants identify populations
  (double neg/pos, single pos).

Fluorescent Dye: 

• FITC (Fluorescein Isothiocyanate)
• green

Key CD Markers for Flow Cytometry:

Most important:

• Pan T → CD3;

• Pan B → CD19;

• Myeloid → MPO;

• Blast → HLADR, TdT, CD34

Solving Flow Cytometry Questions (Phenotyping Leukemias):

Markers	Leukemia Type	Cell Association
TDT+, CD3+	T-ALL	7T-blast
TDT+, CD19+	B-ALL	B-blast
HLADR+, MPO+	AML	Myeloid blast
HLADR+, CD3+, MPO+	Biphenotypic Leukemia	T-cell + myeloid markers

Clinical Applications of Flow Cytometry

• Leukemia typing

• HIV (CD4/CD8 counts)

• Paroxysmal nocturnal hemoglobinuria

• Detection of fetal RBCs in maternal circulation

PCR (Polymerase Chain Reaction)

• An in-vitro amplification technique of desired DNA
• Achieved by designing primers complementary to flanking sequences of desired DNA.

• Generates multiple copies of a DNA fragment.

• It is replication in a test tube.

• Prerequisites
• Thermocycler controls the temperature cycles
• DNA template
• DNA primers
• a heat stable DNA polymerase
• deoxynucleotide triphosphates (dNTPs)
• NOT Dideoxynucleotides
• Needed for Sequencing

Isothermal Amplification Techniques

• Allow all steps at a constant temperature.

• Eliminates the need for a thermocycler.

LAMP assay

• All steps are at a constant 60°C.

• Loop mediated amplification assay

• A simple incubator is sufficient.

• Bacillus stearothermophilus (BST) DNA polymerase is used.
• Also used as a Bioindicator in Autoclaves.

• Bacillus smithii (BSM) DNA Polymerase is another enzyme used.

• Both enzymes have high strand invasion ability.

• This allows DNA unwinding at lower temperatures (60°C).

Types of PCRs:

Microarray

• Also known as Chip technology.

• Used to understand the pathogenesis of a disorder.

• Identifies differences in gene expression between tumour and normal tissue.

Methodology:

• Mnemonic: Micoarray → M for M → mRNA

• mRNAs are extracted and converted to cDNA.

• cDNAs are differentially labelled with fluorescent dyes.
• e.g., red for tumour, green for normal.

• Labelled cDNAs are applied to a microarray chip.

• Each well on the chip contains specific oligonucleotides.

• The cDNAs compete to bind with these oligonucleotides.

Interpretation:

• Yellow fluorescence: Gene is equally expressed.

• Red fluorescence: Gene is overexpressed in tumour tissue.

• Green fluorescence: Gene is not expressed (or underexpressed) in tumour tissue.

• Findings help in choosing a treatment modality.

Comparative Genomic Hybridization (CGH) /
Chromosome Painting

• A cytogenetic version of microarray.

• Used to understand chromosomal aberrations.

• Compares normal genes (green) with patient genes (red)

• Allows for painting all chromosomes

• Mnemonic: C for C → Chromosomes

Methodology:

• Chromosomes from diseased individuals are extracted and digested.

• They are denatured and labeled with a fluorescent dye (e.g., red for tumour DNA).

• Standard (normal) chromosome fragments are labeled with a different dye (e.g., green).

• Both DNA fragment types are mixed and spread on a CGH chip.

Interpretation:

• Yellow fluorescence:
• Segment is equally present in both samples.

• Green color:
• Segment is not in tumour DNA (a microdeletion).
• Loss of function mutation

• Red color:
• Segment is overexpressed in tumour tissue (an amplification).
• Gain of function mutation

Question Discussion:

• Q. The metaphase chromosome in comparative genomic hybridisation painted purple represent:
• A. Balanced DNA content
• B. Amplification in tumour DNA
• C. Under expression in tumour DNA
• D. Overrepresentation of the whole chromosome in tumour DNA
Explanation:
• If purple (tumour DNA color) is seen alone, it indicates amplification.
• If aqua blue (normal DNA color) is seen alone, it indicates a deletion.

Satellite DNA / Highly Repetitive Sequences

• Repeated more than one lakh times in the human genome.

• Distributed across various loci.

• The number of repeats at a locus is unique to each individual.

• Useful for personal identification.

• Classified into:
• Microsatellite repeats
• Minisatellite repeats (VNTR)

• Used in forensic applications.
• Person identification.
• Paternal dispute cases.

Steps in Paternal Dispute Cases

• Step 1: Sample Collection:
• Blood from mother, child, suspected father.

• Step 2: DNA Extraction:
• From all three samples.
• Centrifuge blood for the buffy coat (WBCs).
• Use a high salt method to lyse cells and release DNA.

• Step 3: Analysis of Microsatellite Repeats:
• At least 5 repeats at 5 loci are analysed.
• Example:
• If child's DNA is "8/8".
• If maternal DNA is "11/8", one '8' came from the mother.
• The suspected father must have the other '8' to be the biological father.

Steps in Molecular Diagnostics of known mutation sites

Question Discussion: Molecular Diagnosis Steps

• Q. A 21-year-old man wants a molecular diagnosis of sickle cell anemia... Help him choose appropriate steps and arrange them...
The correct sequence is
• Sample collection, DNA extraction, Conventional PCR, RFLP
• Explanation:
• RT-PCR is for RNA.
• Cytogenetics is for chromosomal aberrations.
• Molecular diagnostics identifies nucleotide sequence differences.

Example:

• Sickle Cell Anemia

Steps

1. Sample Collection:
• Preferred sample is blood.

2. DNA Extraction:



• Centrifuge blood for the buffy coat (WBCs)
• Use a high salt method to lyse cells.
• 6M NaCl
• GITC
• All 46 chromosomes are extracted.

3. PCR (Polymerase Chain Reaction):
• The extracted DNA undergoes PCR.
• Uses primers specific for the beta-globin gene fragment (site of mutation).
• This step amplifies the specific DNA fragment.

4. Detection of Mutation:
• Uses DNA sequencing or RFLP.

Sequencing

• Directly determines if the DNA is normal or abnormal.

• Generally not cost-effective or readily available.

RFLP (Restriction Fragment Length Polymorphism)

• Uses restriction enzymes that cut DNA at specific palindromic sites

• A specific enzyme is chosen that cuts at the mutation site only if normal.

• Diagnosis is based on whether the DNA is cut.

• Applications
• Paternal disputes
• Crime
• Genome mapping

Restriction enzyme

Example 1: Sickle Cell Anemia:

• Primers amplify a 1.4 kb fragment of the beta-globin gene.

• The enzyme Mst II cuts this site only if it is normal.

• Normal (wild-type):
• Mst II cuts the 1.4 kb fragment into 1.2 kb and 0.2 kb fragments.

• Mutated (sickle cell):
• Mst II does not cut; the fragment remains 1.4 kb.

• Fragments are separated by Electrophoresis.

• Interpretation:
• 1.4 kb band: Uncut, mutated (SS genotype).
• 1.2 kb band: Cut, normal (AA genotype).
• Two bands (1.4 kb and 1.2 kb): Carrier (AS genotype).

Example 2: Paternal Dispute / Crime

In a surgical intensive care unit, restriction fragment length polymorphism (RFLP) was used to identify five different staphylococci species. In which of the following situations do restriction enzymes act?

1. TAGATA/ATCTAT

2. ATGGAC/TACGTG

3. AATATA/TATAAT

4. GATTAC/CATTAG
ANS

1. GATTAC/CATTAG

RTPCR vs qPCR

Cytogenetics

• Studies chromosomes to detect chromosomal aberrations.

Types of Cytogenetics Techniques

Karyotyping:

• Karyotyping analysis
• Light microscopy used to visualise chromosomes
• Resolution: 5 mb (Megabases)

• Best technique for detecting monosomy & trisomy

• Uses Giemsa staining of chromosomes.

• Identifies copy number variations (e.g., Trisomy 21).

• Limitation: Cannot detect structural alterations like translocations.

Process

• Chromosomes arranged and photographed to produce a karyogram.

Samples: 

• Chorionic Villus Sampling (CVS) (10 wks); 

• Amniotic fluid (14-18 wks); 

• Blood sample (lymphocytes).

Steps:

1. Arrest cells:
• At Metaphase using Colchicine.

2. Fixation:
• With Carnoy's fixative (Methanol + Glacial Acetic Acid, 3:1 ratio).
• Mnemonic: Kar -Yo ⇔ Car -oy

3. Staining:
• G-banding 
• (Giemsa stain, most common, light microscope)
• Q-banding 
• (Queen banding → ↑ microcsope → fluorescent microscope)

Technique	Description
G banding	Metaphase chromosomes → treated with trypsin (partial denaturation of chromatin) → stained with Giemsa staining It is the most common technique used in cytogenetics to produce a visible karyotype by staining condensed chromosomes. Shows light and dark banding patterns.
Q banding	Fluorescence staining with quinacrine mustard. Requires a UV fluorescence microscope. Produces banding patterns similar to G banding.
R banding	Reverse Giemsa staining. In this technique, the light and dark areas are reversed compared to G banding.
C banding - Stain centromere	Preferentially stains heterochromatin, especially in centromeric regions, using silver salt staining. Helps identify dicentric chromosomes (those with two centromeres).
T banding - Stain telomere	Specifically stains the telomeres (terminal ends) of chromosomes. Useful for analyzing chromosome ends.

Chromosome Arrangement:

• In decreasing order of length.

• Shortest chromosome:
• Chromosome 21.

Limitations:

• Lengthy → Time consuming

• Cells need to be in metaphase for chromosome visualization.

• Limited resolution: Cannot detect:
• Microdeletions
• Amplifications
• Complex translocations

What does the division of a chromosome perpendicular to the normal axis of division lead to?

1. Ring chromosome

2. Isochromosome

3. Acrocentric chromosome

4. Subtelocentric chromosome
ANS

FISH (Fluorescent In situ Hybridization):

• Uses fluorescently-labeled probes.

• Probes are complementary to specific chromosome segments.

• Probes hybridize to metaphase-arrested chromosomes.
• Arrest cells: At Interphase → (Interphase FISH) → faster, doesn’t require cell culture.

• Viewed under a fluorescence microscope.

Applications:

• Copy Number Variations:
• A Trisomy 21 probe shows three signals instead of two.

• Structural Alterations
• Fish for MAT
• NOT Point Mutation
1. Microdeletions
• Probes for suspected deleted regions are used.
• If deleted, no signal appears.
2. AMPLIFICATION
3. Translocations
• For BCR-ABL, two probes with different colors are used.
• In translocation, signals appear on one fused chromosome.

Types of FISH

• Chromosomal painting
• Each chromosome labelled with fluorescent probe
• Not all chromosomes get unique colours (dye limitation)

• Multicolour FISH (Spectral Karyotyping, SKY)
• 23 unique colours (from 5 fluorophores)
• Each chromosome gets unique colour
• Uses:
• Detects structural + numeric abnormalities
• Detects gene amplification + microdeletions
• Maps gene to chromosomal locus
• Limitation: needs prior knowledge of genes & chromosomes

• Interphase FISH
• Rapid, no need of growing cells
• Uses: prenatal diagnosis, tumour diagnosis

Limitations:

• Cannot detect
• point mutations.
• unknown defect
• Requires prior knowledge of the suspected defect.
• Use Comparative Genomic Hybridization (CGH) / Chromosome Painting
• Checks every segment of all 46 chromosomes.
• Provides a signal wherever an aberration is present.

Question Discussion:

• Q. FISH is used to detect all of the following except?
• A. Translocation
• B. Amplification
• C. Deletion
• D. Point mutation
Explanation:
• FISH cannot detect point mutations.
• PCR/RFLP or PCR/Sequencing is used for point mutations.