DNA Basics and Molecular Biology Refresher¶

DNA Structure and the Reverse Complement Function¶

DNA is a double-stranded molecule arranged in an antiparallel fashion, meaning one strand runs in the 5' to 3' direction while its complementary strand runs in the 3' to 5' direction. The two strands are held together by base pairing: Adenine (A) pairs with Thymine (T), and Cytosine (C) pairs with Guanine (G). These fundamental properties allow for key sequence operations like reversing and complementing strands. To understand this structure visually, watch the video below:

Reverse¶

To reverse a DNA sequence, simply write it backward. For example:

Original: 5'- ATGCAG -3'
Reversed: 5'- GACGTA -3'

Complement¶

To complement a DNA sequence, replace each base with its pairing base:

A (Adenine) ↔ T (Thymine)
C (Cytosine) ↔ G (Guanine)

Example:

Original: 5'- ATGCAG -3'
Complement: 5'- TACGTC -3'

Reverse Complement¶

The reverse complement is simply applying the complement rule after reversing the sequence:

Reverse the sequence.
Replace each base with its complementary base.

Example:

Original: 5'- ATGCAG -3'
Step 1 (Reverse): 5'- GACGTA -3'
Step 2 (Complement): 5'- CTGCAT -3'

Since DNA is double-stranded, the reverse complement operation essentially determines what the partner strand would look like. When we take the reverse complement of a sequence, we are simulating the process of reading the opposite strand in the correct 5' to 3' direction, just as it would naturally exist in a DNA molecule. Calculating the reverse complement is a very common operation when working with DNA sequence. However, reverse and complement, individually, are only used in specialized scenarios. It is important you understand what these operations are, but it is mainly so you understand the composite of the two. For the purposes of this tutorial, you will never use individual reverse or complement operations again.

Try it Yourself¶

Try calculating the Reverse, Complement, and Reverse Complement of these sequences:

1️⃣ Reverse

What is the reverse of 5'- AGCTTG -3'?

2️⃣ Complement

What is the complement of 5'- AGCTTG -3'?

3️⃣ Reverse Complement

What is the reverse complement of 5'- CAGGTAG -3'?

The Central Dogma¶

The Central Dogma of molecular biology describes 3 biochemical processes happening to biomolecules in most cells. During Replication, an exact copy of DNA is produced. During Transcription, a region of DNA is read into RNA. During Translation, a region of RNA (an mRNA or messenger RNA) is read into protein. Though these processes are very complex and involve numerous biomolecules, the sequences of RNA and protein are readily predictable from the DNA sequence.

Transcription¶

A gene refers to a region of DNA that encodes a protein. You can think of it like a song on a vinyl record—there is a physical region to the gene, with clear starts and ends. A gene can be on either strand of DNA, but it is always flanked by a promoter and a terminator sequence. The promoter determines which strand will be the "coding strand," as the RNA polymerase binds to the promoter and proceeds until it reaches the terminator.

During transcription, an RNA molecule is created that is identical to the coding strand but with Uracil (U) replacing Thymine (T).

Example:

DNA Coding Strand: 5'- ATGCAGTAC -3'
RNA Transcript: 5'- AUGCAGUAC -3'

Quiz: Predict the RNA Sequence¶

What is the RNA sequence transcribed from this DNA coding strand?

5'- TCTGACTA -3'

Translation¶

An mRNA transcript consists of a 5' UTR (Untranslated Region) followed by the CDS (Coding DNA Sequence). The CDS is an ORF (Open Reading Frame), meaning a sequence that, when read in groups of three bases (codons), begins with a start codon (ATG, GTG, or TTG) and ends with a stop codon (TAA, TAG, TGA).

The ribosome recognizes the Shine-Dalgarno sequence and the start codon, then reads the RNA three bases at a time, translating each codon into an amino acid based on the genetic code.

Genetic Code Table¶

Note: While translation occurs from an RNA sequence, for convenience, we often refer to codons in their DNA format. Codons are three-letter sequences that specify amino acids during protein synthesis. Each codon in DNA corresponds to an RNA codon by replacing Thymine (T) with Uracil (U).

Codon	Single Letter	Full Name	Codon	Single Letter	Full Name
TTT	F	Phe	TTC	F	Phe
TTA	L	Leu	TTG	L	Leu
TCT	S	Ser	TCC	S	Ser
TCA	S	Ser	TCG	S	Ser
TAT	Y	Tyr	TAC	Y	Tyr
TAA	stop	Ter	TAG	stop	Ter
TGT	C	Cys	TGC	C	Cys
TGA	stop	Ter	TGG	W	Trp
CTT	L	Leu	CTC	L	Leu
CTA	L	Leu	CTG	L	Leu
CCT	P	Pro	CCC	P	Pro
CCA	P	Pro	CCG	P	Pro
CAT	H	His	CAC	H	His
CAA	Q	Gln	CAG	Q	Gln
CGT	R	Arg	CGC	R	Arg
CGA	R	Arg	CGG	R	Arg
ATT	I	Ile	ATC	I	Ile
ATA	I	Ile	ATG	M	Met
ACT	T	Thr	ACC	T	Thr
ACA	T	Thr	ACG	T	Thr
AAT	N	Asn	AAC	N	Asn
AAA	K	Lys	AAG	K	Lys
AGT	S	Ser	AGC	S	Ser
AGA	R	Arg	AGG	R	Arg
GTT	V	Val	GTC	V	Val
GTA	V	Val	GTG	V	Val
GCT	A	Ala	GCC	A	Ala
GCA	A	Ala	GCG	A	Ala
GAT	D	Asp	GAC	D	Asp
GAA	E	Glu	GAG	E	Glu
GGT	G	Gly	GGC	G	Gly
GGA	G	Gly	GGG	G	Gly

Quiz: Translate the DNA Sequence¶

What is the amino acid sequence for the following DNA sequence?
5'- ATG CAG GTA GAA TAA -3'

Summary of Terms¶

Fundamental DNA Concepts¶

DNA (Deoxyribonucleic Acid): A double-stranded molecule that stores genetic information in all living organisms.
Base Pairing: The specific hydrogen bonding between Adenine (A) & Thymine (T) and Cytosine (C) & Guanine (G) in DNA.
Antiparallel: The orientation of DNA strands where one runs 5' to 3' and the complementary strand runs 3' to 5'.

Transcription & RNA Concepts¶

RNA (Ribonucleic Acid): A single-stranded nucleic acid transcribed from DNA, involved in protein synthesis.
Transcription: The process where RNA polymerase synthesizes RNA from a DNA template.
RNA Polymerase: The enzyme that transcribes DNA into RNA.
mRNA (Messenger RNA): The RNA transcript that carries genetic information from DNA to ribosomes.
5' UTR (Untranslated Region): The non-coding region at the start of an mRNA that regulates translation.
3' UTR (Untranslated Region): The non-coding region at the end of an mRNA that influences stability and translation efficiency.
Promoter: A DNA sequence where RNA polymerase binds to start transcription.
Terminator: A DNA sequence signaling the end of transcription.

Translation & Protein Synthesis¶

Translation: The process where ribosomes synthesize proteins by reading mRNA codons.
Ribosome: The molecular machine that assembles proteins by reading mRNA codons.
tRNA (Transfer RNA): An RNA molecule that brings amino acids to the ribosome during translation.
Codon: A three-nucleotide sequence in DNA or RNA that specifies an amino acid or stop signal.
Anticodon: A complementary three-base sequence on tRNA that pairs with an mRNA codon.
Start Codon: The first codon in an mRNA sequence that signals the beginning of translation (ATG coding for Methionine).
Stop Codon: A codon (TAA, TAG, TGA) that signals the termination of translation.

Gene Structure & Regulation¶

Gene: A specific region of DNA that encodes a functional product, usually a protein.
CDS (Coding DNA Sequence): The protein-coding portion of a gene within an mRNA.
Open Reading Frame (ORF): A sequence of codons beginning with a start codon and ending with a stop codon.
Ribosome Binding Site (RBS): A region on mRNA where the ribosome binds to initiate translation. In prokaryotes, this includes the Shine-Dalgarno sequence, while in eukaryotes, it is often the Kozak sequence.
Shine-Dalgarno Sequence (SD): A ribosomal binding site in bacterial mRNA, located upstream of the start codon, which aligns the ribosome for efficient translation initiation.
Kozak Sequence: A sequence surrounding the start codon in eukaryotic mRNA that enhances ribosome recognition and translation efficiency.