Molecular Biology 101 for Techies

Many recent breakthroughs in cancer therapy fall into the category of personalized medicine. Cancers are characterized more deeply using Molecular Pathology. Another revolutionizing technology that opens up new possibilities to researchers is spatial biology, including its sub-disciplines proteomics and spatial transcriptomics. When reading about these technologies, one is faced with terminology that is far from native to many data scientists who enter this field with a technical background. And that’s where our Molecular Biology 101 for Techies comes in.

To give an example: We are interested in learning how methods such as bulk or single-cell sequencing work. They can sequence the genome, exome, or individual gene transcripts. Along the way, processes such as permeabilization are required and on a microscopic scale, so-called oligos are hybridized to complementary DNA strands and sometimes carry multiple fluorophores that form a unique barcode.

This article will give a brief overview about a range of molecular biology basics. If you are more interested, how these basics are applied in real-world research, read our Spatial Biology 101.

Biological glossary

In the following, we try to collect some of the frequently used vocabulary, and try to organize and explain it without requiring any deep biological prior knowledge.

Infographic of a biological glossary in our Molecular Biology 101

Let’s begin with a biological glossary of the most relevant basic elements:

Nucleobases (“bases”)Nitrogen-containing base
DNA strands are made of 4 nucleobases:
ACGT (adenine, cytosine, guanine, thymine)
RNA strands are made of 4 nucleobases:
AGCU (uracil instead of thymine)
A DNA molecule is composed of two DNA
strands held together by hydrogen bonds
between the complementary paired bases.
The three-dimensional structure of DNA
is a double helix.
NucleotideNucleotides are composed of a nucleobase,
a five-carbon sugar and one or more phosphate groups.
In DNA, the sugar is deoxyribose. In RNA, the sugar is ribose.
The abbreviation is “nt”.
Oligonucleotides are sequence of nucleotides,
typically created synthetically in a lab.
They are used as “probes” for specific DNA or RNA
sequences e.g. in  DNA microarrays, FISH,
Southern blots, PCR.
Naming convention: ends with –mer.
Examples: 6x nt = Hexamer, 20x nt= 20-mer
GeneRegion of DNA that is transcribed as a single
protein or a single RNA.
Sequence of nucleotides, in the range of
1.000 – 1.000.000 base pairs
GenomeEntire set of DNA genes belonging to an organism.
Comprises ca 20.000 -25.000 genes
GeneticsStudy of the genes of an organism on the
basis of heredity and variation
EpigeneticsStudy of phenotypic changes in organisms
caused by modification of gene expression
ExonsSubset of a DNA sequence that encode RNA.
DNA is made up of exons and introns.
When it is translated into RNA, only the exons
are kept, while introns are discarded.
IntronsSubset of a DNA sequence that does not become
part of the final RNA product.
ExomeSet of all exons, i.e., protein-coding subset of the genome.
The whole exome refers to the portion of the
human genome that encodes for proteins, known as exons.
The exome makes up approximately 1-2 % of the human
genome and includes the coding regions of all genes.
TranscriptomeAll of the RNA molecules that are produced by the cell’s genes.
The term “transcriptome” is sometimes used ambiguously:
either the entire RNA or only the coding mRNA is meant.
·       Coding mRNA: 1-4 % of the RNA – codes for proteins
·       Non-coding: does not give rise to proteins
The size of transcriptome varies heavily per cell type
ProteinLarge molecules comprised of amino acids that
perform various functions in the cell.
ProteomeAll proteins of a cell or a cell compartment that are
expressed under precisely defined conditions
and at a specific time (~ 1 Mio proteins)
Codon“three-letter word” formed by 3 nucleotides (“bases”)
that codes an amino acid, e.g. TTT, ACT, CAG ,…
would represent three amino acids.
Amino Acidsbuild blocks for proteins. Proteins are made of
only 20 amino acids linked together in chains (“polypeptides”)


Meaning“The human code”
Gentetic code —
the entire genetic information
of a organism is
stored in the DNA.
Nearly all human cells
contain the same DNA.
So more insightful to
look at RNA or proteins.
Responsible for protein synthesis;
RNA present in cells varies
in quantity and type.
FormDouble-helixSingle strand, folded onto itself
StabilityRelatively stableRelatively unstable
Nucleobases4: A, C, G, T4: A, C, G, U
Ribose has a hydroxyl group,
which makes RNA more chemically labile
SubtypesProtein-coding subset
of genome is called exome.
mRNA, tRNA, rRNA, snRNAs, non-coding RNA
(“non-coding” means here that this RNA
type is not involved in protein synthesis)
ProcessesExons are transcribed to mRNAmRNA delivered to ribosomes
(factories in the cytoplasm).
Ribosomes read message of mRNA (“recipe”) and assemble amino
acids into proteins.

History excursion: Discovery of the DNA double helix

British physicists and molecular biologists James Watson (left) and Francis Cricks (right) present their model of the DNA double helix
British physicists and molecular biologists James Watson (left) and Francis Cricks (right) present their model of the DNA double helix (© Christie’s Images/​Reuters)

British physicists and molecular biologists James Watson and Francis Cricks discovered the molecular structure of DNA. For a few years then, the “race for the DNA structure” had been going on, which they won with their publication in Nature on April 25th in 1953. Their paper was only one page long and contained merely 6 references. They combined cues from their own work as well as unpublished literature, notably the famous “photo 51” from their rivaling King’s College London colleague Rosalind Franklin. They had the break-through idea, that the structure must be a double-helix (not a triple-helix as previously falsely postulated by chemist Linus Pauling) where the bases are directed inwards, not outwards. Posed then with the problem that the 4 bases do not have the same sizes and forces and in line with the — at the time well known — observation that two pairs of bases were suspciously always observed at equal amounts, they figured out that not identical but rather complimentary bases interconnected the two leixes (via a hydrogen bond):  adenine pairs with thymine, and cytosine pairs with guanine. Watson and Cricks, together with Maurice Wilkins, who pioneered the X-ray diffraction method, later received the Nobel price. Rosalind Franklin, who died only 3 years before from cancer (not unlikely the result of exposion to X-rays in her research), was not honored.

Genotype vs. Phenotype

GenotypeThe genotype refers to the genetic information
that an individual inherits from its parents.
PhenotypeThe phenotype of an organism refers to its
observable characteristics or traits,
such as physical appearance, behavior,
and biochemistry.

“phenotype = genotype + environment”


By now, a few patterns may have become obvious

  • Genomics is the large-scale study of the genome (or part thereof). The genome is the collection of all genes.
  • Transcriptomics is the art of measuring the transcriptome (or part thereof), which is the collection of all transcripts that are produced by a cell’s genes.
  • Proteomics is the large-scale study of the proteome (or part thereof), which is the collection of all proteins.

Biological processes involved in sequencing

Different technologies can be used for measuring the genome / transcriptome / proteome. These methods involve various biological processes:

SynthesisRNA synthesis: catalyzed by an enzyme:
RNA polymerase
PermeabilizationMaking cell membrane permeable so that
DNA/RNA can get out
HybridizationBinding of complementary strand to
a target strand
Replication  DNA is clonally amplified to more DNA,
e.g., with PCR Enzyme: DNA polymerase
TranscriptionDNA is transcribed to mRNA (Process of
copying a segment of DNA into RNA)
Enzyme: RNA polymerase
TranslationRNA is translated to a protein


“Thanks” to the Covid pandemic, everyone has heard of PCR. But is a PCR used to measure genes? Not really. It can rather be regarded as a pre-processing step that is required to duplicate the DNA in a small sample to the level that it becomes measurable by sequencing technologies. PCR stands for polymerase chain reaction. The original DNA sample to be copied is called the “template”.

PCR devices perform a sequence of steps in multiple cycles.

  1. In each cycle, the device first heats up the sample in order to break up the DNA double-helix into two single strands.
  2. Next, short oligos called “primers” bind to the now single-stranded DNA.
  3. Then the polymerase enzyme triggers a process called polymerization, in which free-floating T, C, G, or A bases successively bind to one side of the primer. In each turn, only the base that is complementary to the base in the DNA template can attach to the primer and the already bound bases. Base by base, the DNA strands are complemented and new double-helixes are formed.

The steps 1-3 are repeated over and over. In each cycle, the number of DNA strands is approximately doubled.


After the sample has been amplified using PCR, it can be sequenced.

Various types of sequencing exist. The term Next Generation Sequencing (NGS) may be familiar to everyone. But how many generations of sequencing were there before NGS you may ask. Next Gen Sequencing is actually only the 2nd generation, the first one being Sanger Sequencing invented in 1977. It has been used for four decades and was then replaced by NGS, where devices became commercially available in ca. 2005. First generation sequencing suffered from very low throughput. NGS solved this challenge by employing massively parallel sequencing, typically in flow cells. NGS can read hundreds of megabases to gigabases of nucleotide sequence reads in a single instrument run. The market leader for NGS is San Diego based Illumina Inc. Briefly, they use an approach called Sequencing by synthesis, in which a fluorescently labelled base emits light when it is incorporated into a growing DNA. This light is then imaged with a microscope and analyzed.

RNA-Sequencing, abbreviated RNA-seq, refers to the sequencing of RNA instead of DNA. Many devices first reverse-transcribe RNA to “complementary DNA” (cDNA), which is then in turn sequenced with short-read sequencers like Illumina’s HiSeq. A drawback of this approach is that errors are introduced during the reverse transcription. Other devices like NanoString’s nCounter instead measure the RNA directly and do not rely on using cDNA as a proxy for the RNA. So does, real-time RNA sequencing such as the “USB stick” solution by Oxford Nanopore Technologies. In real-time sequencing, bases are reported while the sequencing is still ongoing.

Number of sequenced genes or transcripts

When a technology allows measuring only a limited set of genes, it falls into the category of Targeted Sequencing. Oppositely, when all genes can be distinguished, this is referred to as Whole-Genome-Sequencing.

Whole-genome sequencing
Method can sequence all genes
Whole-exome sequencing
Method can sequence all exons
(protein coding regiosn of the DNA)
Whole-transcriptome sequencingMethod can sequence all
transcripts (RNA)

Sequencing: Bulk → Single Cell → Spatial

Bulk-Sequencing means that the RNA or DNA from many cells is “pooled”. While this sequencing method is simpler, its obvious drawback is that the genes from many cells are measured and so the result may get “polluted” by non-target cells that were merely “bycatch”. For instance in clinical molecular pathology, the intention is usually to sequence only tumor cells in order to identify the tumor subtype and decide which therapy to apply. Cells are “scraped” from the (supposed) tumor area in the tissue and so care has to be taken that this set of cells contains a large portion of tumor cells.

The opposite of bulk sequencing is Single Cell Sequencing, abbreviated “scSeq”. Like in bulk sequencing a pool of cells is first collected, but in a preprocessing step, a device first labels each cell with a unique molecular barcode. The 10x Genomics Chromium Controller, for instance, does this by encapsulating a single cell together with reagents in a droplet and stimulating a micro reaction that leads to the barcoding. Other technologies separate individual cells into micro-wells instead of droplets. After each cell has been barcoded, the regular bulk-sequencing can be carried out. Thanks to the unique barcodes, transcripts that stem from the same cell can be identified and grouped. The next evolution is Spatial Sequencing, where transcripts cannot only be grouped by cell, but where the origin of the cell in the tissue is known. This way, both the morphology and genome can be examined at the same time and the cell’s environment, i.e., its neighbor cells, are also known. 

Alternative Sequencing Methods

Fluorescence in situ Hybridization (FISH) is a spatial sequencing technology. Oftentimes, only a very limited (often single-digit) number of genetic events is detected. For instance, FISH HER2/neu assays employ only two probes that visualize HER2 and CEP17 genes, so that their ratio per cell can be calculated in order to determine if a HER2 overexpression exists. If it does, then this is the driver for the tumor. This companion diagnostic test then indicates a targeted therapy with a HER2 inhibitor (“blocker”) such as Trastuzumab.  

quantitative PCR (qPCR), sometimes also called real-time PCR, is a variant of PCR that quantitatively reports in real-time the amplification of a DNA molecule. It can be regarded as a targeted sequencing method. The principle of qPCR is that a fluorophore is added to the primer, which is then imaged and measured. The output is the relative gene expression (or mRNA copy number).

download MIKAIA for free from

Avatar photo

Volker Bruns

Volker is a digital pathology and spatial biology enthusiast with a computer science background. Volker and his team develop commercial image analysis software for digital pathology and offer contract development, as well as image analysis as a service in the life sciences.

Add comment

Get started now

Download MIKAIA for free from

Don’t miss any news

Sign up now for the MIKAIA newsletter

Get in touch with us

Questions, remarks, feature
requests, project inquiries, …
email us:

Dr. Volker Bruns
Group Manager
Medical Image Analysis (MIA)
Digital Health and Analytics | Fraunhofer IIS