This document discusses molecular genetic diagnosis techniques. It begins by describing how techniques like karyotyping, Southern blotting, and Sanger sequencing revolutionized genetic diagnosis in the late 20th century. It then covers the various indications for genetic testing, including inherited conditions, prenatal testing, and acquired conditions like cancer. The rest of the document details specific molecular analysis techniques used, such as PCR, Sanger sequencing, pyrosequencing, restriction fragment length analysis, FISH, MLPA, Southern blotting, and next generation sequencing. It provides examples of the medical applications of these various techniques.

1. MOLECULARGENETICDIAGNOSIS
Dr.T.Arivazhagan
Post Graduate
Dept. Of Pathology

3. Introduction
• In the later half of 20th century they used conventional karyotyping
For diagnosis of cytogenetic disorders
• Ex: down syndrome
• DNA based assays
• Southern blot – Huntington disease
• Over a period of time
• Sanger DNA sequencing in 1977
• PCR in 1983
• These 2 methods allowed routine sequencing of any known segment of DNA
• Today nucleic acid based testing take a central role in the genetic diagnosis

4. Indications
1. Inherited
2. Acquired

5. Inherited
•It is required at an age , depending on the clinical
presentation
•During prenatal / post natal / childhood / older age group
•Most recessive pattern – have limited number of recurrent
mutations
•Dominant pattern – mutations scattered throughout the
responsible gene.

6. Prenatal indications
• Advanced maternal age
• Parent known to have balanced chromosomal rearrangements
• Fetal anomalies observed on ultrasound
• Routine maternal screening ( down syndrome)
• Cystic fibrosis
• Spinal muscular atrophy
• At present usually performed cells obtained by amniocentesis, chorionic villus
biopsy , umbilical cord blood

7. Post natal indications
• Multiple congenital anomalies
• Suspicion of metabolic syndrome
• Unexplained mental retardation
• Suspected aneuploidy
• Suspected monogenic disease

8. Older patients
• Inherited cancer syndrome
• Atypically mid monogenic disease
• Neurodegenerative disorders ( Familial Alzheimer disease, Huntington
disease)

9. Acquired conditions
• Diagnosis & management of cancer:
• Detection of tumor specific mutations
• Determination of clonality
• Identification of specific genetic alterations
• Determination of treatment efficacy
• Detection of drug resistant secondary malignancy
• Diagnosis & management of infectious disease:
• Detection of micro organism specific genetic material
• Identification of specific genetic alteration in the microbes
• Determination of treatment efficacy

10. Tests
• PCR & Detection of DNA sequence alterations:
• Sanger sequencing
• Pyro sequencing
• Single based primer extensions
• Restriction fragment length analysis
• Amplicon length analysis
• Real time PCR

11. Molecular analysis
• Fluorescence in situ hybridization
• Multiplex ligation dependent probe amplification
• Southern blot
• Cytogenetic array technology
• Array based comparative genomic hybridization
• SNP genotyping arrays

12. • RNA analysis
• Next generation sequencing
• Bioinformatics
• Clinical applications
• Targeted sequencing
• Whole exome sequencing
• Whole genome sequencing

13. PCR
• PCR is a technique used in molecular biology to amplify a single copy
or a few copies of a segment of DNA across several orders of
magnitude , generating thousands to millions of copies of a particular
DNA sequence.
• Develop in 1983 by kary mullis
• Using appropriate heat stable DNA polymerase ,Thermal cycling
• The target DNA ( less than 1000 base pairs) are amplified.

15. Sanger sequencing
• Amplified DNA is mixed with DNA polymerase , DNA primer, Nucleotides , 4
dead end nucleotides labelled with different fluorescent tags.
• Reaction produce series of DNA molecules of possible length
• The size are separated by capillary electrophoresis
• The exact sequence can be read & compared with normal sequence to detect
the presence of the mutations.
• Particularly used when analysis of large genes or multiple genes is required.
• This is consider gold standard for sequencing determination

17. Pyrosequencing
• Advantage of release of pyrophosphate when nucleotide is incorporated into a
growing DNA strand.
• Similar to sanger sequencing except instead of terminator nucleotides one at a time
• When one or more nucleotides are incorporated into the growing strands
• The pyrophosphate released and participated in a secondary reaction
• That produce light
• Measured by photo detector.
• More sensitive than sanger sequencing for detection of as little as 5% mutated alleles
present in the background of normal alleles.

19. Single base primer extension
• Useful for identifying mutation at a specific nucleotide position
• An interrogating sequencing primer is added to the PCR product
• Binds only one base upstream of the target
• Differentially coloured terminator fluorescent nucleotides also used
• Very sensitive to detect 1-2% of mutated alleles.
• Disadvantage is only producing one base pair of sequencing data

21. Restriction fragment length analysis
• Advantage of digestion of DNA with endo nucleases that recognize
and cut DNA at specific sequences.
• Amplified PCR product may be digested and normal and mutated
PCR products yield different size fragments
• These different bands are identified by electrophoresis
• Less comprehensive than other methods

22. Amplicon length analysis
• Here 2 primers that flank the region containing trinucleotide repeats at
5’end
• Amplification done
• Large difference in the number of repeats from the normal individual and
mutated individual
• This is easily distinguished by gel electrophoresis

23. Real time PCR
• Variety of PCR based technologies
• Use fluorophore indicators can detect and quantify the presence of
particular nucleic acid sequences in real time
• Used to monitor the frequency of cancer cells bearing characteristic
genetic lesion
• Also used to detect the somatic point mutation in oncogenes
• Advantage is avoiding the need for post PCR analysis

25. MOLECULAR
ANALYSIS OF
GENOMIC
ALTERATIONS

26. FISH
• It is a molecular cytogenetic technique
• Here fluorescent probes are used that bind to only those parts of the
chromosome with a high degree of sequence complementarity.
• It was first developed early 1980’s
• Used to detect & localize the presence or absence of specific DNA sequence

27. • Fluorescence microscopy can be used to find out where the probe is bound to
the chromosome
• It is often used for finding specific features in DNA for use in genetic
counselling, medicine & species identification
• Also used to detect & localize specific RNA targets (mRNA,miRNA) in cells,
circulating tumor cells, tissue samples.

29. Samples
•Prenatal
•Peripheral blood cells
•Imprint smear from cancer biopsies
•Fixed tissue sections

32. Types
• Single-molecule RNA FISH
• Multiple short single labelled fluorescence probe are used
• Fiber FISH
• Chromosomes are attached to a slide in such a way that they are stretched out in a
straight line, rather than being tightly coiled, as in conventional FISH
• Q-FISH
• FISH with PNA’s with computer software to quantify the result
• Used in telomere length research

33. •Flow-FISH
• Flow cytometry used
•MA-FISH
• Microfluidic flow to increase DNA hybridization efficiency
• Detecting the HER2 gene in breast cancer tissues

34. Medical applications
• Prader-Willi syndrome
• Angelman syndrome
• 22q13 deletion syndrome
• Chronic myelogenous leukemia
• Acute lymphoblastic leukemia
• Velocardiofacial syndrome
• Down syndrome
• Sperm cells to find somatic or meiotic karyotype
• Infection – suspected micro organisms

35. MULTIPLEX LIGATION DEPENDENT
PROBE AMPLIFICATION
• It is a variation of the multiplex polymerase chain reaction that
permits amplification of multiple targets with only a
single primer pair
• Used to detect deletions & duplications of any size
• Including anomalies that too large detected by PCR & too small
detected by FISH

37. Uses
• Detection of mutations and single nucleotide polymorphisms
• Analysis of DNA methylation
• Relative mRNA quantification
• Chromosomal characterization of cell lines and tissue samples
• Detection of gene copy number
• Detection of duplications and deletions in human cancer predisposition genes such
as BRCA1, BRCA2, hMLH1 and hMSH2 and
• Aneuploidy determination.
• MLPA has potential application in prenatal diagnosis both invasive and noninvasive

38. Southern blotting
• Detection of a specific DNA sequence in DNA samples
• Hybridization of radiolabeled sequence specific probes to genomic DNA
• This has been digested by restriction enzyme
• Separated by gel electrophoresis
• Its rarely used but remains useful in detection of large trinucleotide expansion
disease

39. Next generation sequencing
• The term used to describe several newer DNA sequencing
technologies that are capable of producing large amount of sequence
data in a massively parallel manner
• Differ from sanger sequencing by its input sample requirements
• In sanger – Single simple homogenous template DNA needed
• In NGS – No need of such material
• Any DNA from almost any source can be used.

40. Basic process
•Spatial separation
•Local amplification
•Parallel sequencing

42. Types
• Targeted sequencing
• Only targeted gene or panel of gene used to minimize the sequence cost
• Whole exome sequencing
• Type of targeted sequencing
• Used to detect protein coding mutations
• Whole genome sequencing
• When exome sequencing gives negative result but still clinical suspicion is
still high