Institute of Human Genetics

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Molecular Genetics


Study in molecular genetics involves investigations based on DNA or RNA and any change in the normal genome sequence is identified by amplification of the specific DNA segment of interest. This can be achieved by several molecular techniques and most common are PCR.

What is is PCR (Polymerase chain reaction)? 

PCR is a biotechnological tool to analyze a short sequence of DNA (or RNA) in samples containing only minute quantities of DNA or RNA. It is used to amplify multiple copies of selected region of DNA or RNA. The technique is highly efficient where untold numbers of copies can be made of the DNA. Moreover, PCR uses the same molecules that nature uses for copying DNA: Any PCR reaction includes

  • Two "primers", short single-stranded DNA sequences that are synthesized to correspond to the beginning and ending of the DNA stretch to be copied;
  • An enzyme called Taq polymerase that moves along the segment of DNA, reading its code and assembling a copy; and
  • A pile of DNA building blocks (dNTPs) that the polymerase needs to make that copy.

For any point mutation or repeat copies PCR is the standard tool to use for mutation detection.


What is Real Time PCR (RT PCR)?

Real-time polymerase chain reaction (RT PCR) is also called as quantitative polymerase chain reaction (qPCR), a technique of molecular biology based on the polymerase chain reaction (PCR), which is used to amplify and simultaneously quantify a targeted DNA molecule. For one or more specific sequences in a DNA sample, quantitative PCR enables both detection and quantification. The quantity can be either an absolute number of DNA copies or a relative amount when normalized to DNA input or additional normalizing genes.

The procedure follows the general principle of polymerase chain reaction; with the key feature of DNA amplification which is detected as the reaction progresses in "real time". This is an advanced approach compared to standard PCR, where the product of the reaction is detected at its end. Two common methods for the detection of products in quantitative PCR are:

(1) non-specific fluorescent dyes that intercalate with any double-stranded DNA, and

(2) sequence-specific DNA probes consisting of oligonucleotides that are labeled with a fluorescent reporter which permits detection only after hybridization of the probe with its complementary sequence to quantify messenger RNA (mRNA) and non-coding RNA in cells or tissues.

The acronym "RT-PCR" commonly denotes reverse transcription polymerase chain reaction and not real-time PCR.

This technique is used to quantify the gene dosage especially to detect the deletion or duplication of the specific gene sequence.


What is Next Generation Sequencing (NGS)?

Next generation sequencing (NGS) is a rapid throughput technology exploding up to > 1 tera bases =1012 achieved by massive parallel sequencing based on the principle of “sequencing-by-synthesis”. This means that the complementary integration of a nucleotide during chain prolongation is directly monitored by the sequencing machine. Remarkably, the increasing sequencing capacity is paralleled by dramatically decreasing costs to sequence a human genome to nearly “$1000 genome”. This will be in the range of those of a magnetic resonance imaging scan.

NGS became available in 2008–09 and the process of applying NGS in a research or diagnostic setting comprises a wet laboratory workflow, including library preparation and the actual sequencing of the library. This is followed by a dry laboratory workflow involving informatics (translation of light signals or pH changes into sequence information) and bioinformatics analyses (sequence alignment, variant calling), as well as variant filtering and interpretation (annotation, mapping against variant databases).

Work flow of a next generation sequencing (NGS) analysis.


With genome sequencing by NGS, about 4 million variants and exome sequencing (covering mainly the 1 % protein-coding part of the genome) results in about 20,000 variants per individual can be detected. It is not only useful in large extended families, where linkage information provides information about the disease locus, but can also be applied to detect disease-causing de novo mutations in sporadic patients.