Introduction to Bioinformatics and Molecular Biology Concepts

Bioinformatics

Bioinformatics involves creating and using computer tools to handle and understand biological and health data.

Scope of Data

The scope of data ranges from millions (10^6) to sextillions (10^21).

Bit

1 piece of information (either a 1 or 0 in binary).

Byte

8 bits (allows 2^8 [256] characters).

Medical Research

Analyzing genomic and imaging data to identify disease markers, develop personalized treatments, and improve diagnostic accuracy.

Behavioral Data

Data from social media, surveys, and observational studies to study human behavior, social trends, and psychological patterns.

Central Dogma of Molecular Biology

The central dogma of molecular biology is DNA → RNA → Protein.

Transcription

The synthesis of RNA from a DNA template.

Translation

The synthesis of proteins based on the sequence of the RNA.

DNA Structure

DNA consists of two strands forming a double helix, with building blocks of phosphate group, deoxyribose sugar, and nitrogen bases (A, T, C, G).

RNA Structure

RNA is single-stranded but can form secondary structures, with building blocks of phosphate group, ribose sugar, and nitrogen bases (A, U, C, G).

Protein Structure

Proteins are made up of amino acids (20 different ones) linked by peptide bonds, with structures including primary, secondary, tertiary, and quaternary.

Genetic Code

The genetic code is a set of rules by which information encoded in DNA or RNA sequences is translated into proteins by living cells.

Start Codons

Start codons (AUG for methionine) signal the beginning of protein synthesis.

Stop Codons

Stop codons (UAA, UAG, UGA) signal the end of protein synthesis.

Codon Table

A codon table is used to determine the amino acid sequence from an mRNA sequence.

Open Reading Frame (ORF)

An open reading frame (ORF) is a part of the sequence that potentially ends up being translated into a protein.

Reading Frames

There are three reading frames in a sequence.

Hydrophobic Amino Acids

Hydrophobic (Nonpolar) amino acids like valine, leucine, and phenylalanine tend to avoid water and stabilize protein structure.

Hydrophilic Amino Acids

Hydrophilic (Polar) amino acids like serine, threonine, and asparagine interact well with water and are often found on the surface of proteins.

Charged Amino Acids

Positive and Negative Charged amino acids can form ionic bonds and are involved in binding oppositely charged molecules.

Protein Folding

Hydrophobic amino acids tend to cluster inside, while hydrophilic amino acids are exposed to the aqueous environment.

Active Sites

The chemical nature and charge of amino acids in the active sites of enzymes are critical for substrate binding and catalysis.

Primary Structure

Amino acid sequence.

Secondary Structure

Backbone interactions, no side chains, any protein can form these, sequence doesn't matter.

Tertiary Structure

Backbone + side chain interactions, more complex structure.

Quaternary Structure

Multiple polypeptides combined.

InDel Mutation

Addition or removal of one or more nucleotides from the DNA sequence.

Frameshift Mutation

Can alter the reading frame of a gene, causing a protein to be nonfunctional or function differently.

Point Mutation

Single nucleotide base change.

Silent Mutation

Change doesn't affect the amino acid sequence of a protein.

Missense Mutation

Change results in a different amino acid being incorporated into the protein.

Nonsense Mutation

Change creates a stop codon, leading to premature termination of the protein.

Impact of Silent Mutations

No effect on protein function because the amino acid sequence remains unchanged.

Impact of Missense Mutations

Can alter the protein's function depending on the properties of the new amino acid and its position in the protein.

Impact of Nonsense Mutations

Usually result in a nonfunctional protein because the translation process is prematurely terminated.

Sickle Cell Anemia

A genetic blood disorder characterized by the production of abnormal hemoglobin (HbS), causing red blood cells to assume a sickle shape.

Glutamic Acid to Valine Substitution

The mutation involves a glutamic acid (Glu) to valine (Val) substitution at position 6 in the β-globin gene.

Charge Difference in HbS

This substitution reduces the overall negative charge, making HbS less mobile in electrophoresis.

Gene Structure

A gene is a section of DNA that contains instructions for making a protein, including regulatory regions, a coding region, and a terminator.

Alternative Splicing

Allows a single gene to produce different protein versions depending on the needs of the cell.

GenBank

The primary database, housing raw sequence data.

RefSeq

Refines genetic records into high-quality reference sequences.

UniProtKB

Specializes in protein functionality, building curated annotations.

Nonredundant Nucleotide Database

Optimizes searches by eliminating duplicate sequences.

RefSeq Information

Provides a curated, standardized set of wild-type sequences, including DNA, RNA, and protein records that have been manually reviewed.

UniProtKB Focus

Focuses on proteins, compiling functional annotations and includes translated nucleotide sequences with supporting literature references.

Nonredundant Nucleotide Database Purpose

Streamlines search efficiency by reducing identical sequences found in GenBank, retaining a single representative entry per unique sequence.

Primary Database

Stores raw DNA and RNA sequence data submitted by researchers, serving as the foundational database for other genetic resources.

Substitution matrix

A substitution matrix is a table used to score alignments between sequences by assigning values to substitutions of one amino acid or nucleotide for another. It helps in quantifying the similarity between sequences.

PAM matrices

PAM (Point Accepted Mutation) matrices are based on evolutionary models and assume knowledge of ancestral sequences.

BLOSUM matrices

BLOSUM (BLOcks SUbstitution Matrix) matrices are derived from observed substitutions in conserved regions of proteins without assuming ancestral sequences.

BLOSUM62 matrix

BLOSUM matrices were developed by analyzing blocks of conserved sequences in protein families. The number in BLOSUM62, for example, indicates that the matrix was derived from sequences with at least 62% similarity.

Probability ratio in substitution matrices

The probability ratio compares the likelihood of a particular substitution occurring in an alignment to the likelihood of it occurring by chance. It helps in scoring alignments.

Calculating sequence similarity

To calculate sequence similarity, align the sequences and use the substitution matrix to score each aligned pair of residues. Sum the scores to get the overall similarity score.

Development process of BLOSUM62 matrix

The BLOSUM62 matrix was developed by aligning conserved blocks of protein sequences with at least 62% similarity, counting the observed substitutions, and calculating the log-odds scores for each substitution.

Goal of sequence alignment

The goal is to arrange sequences to identify regions of similarity that may indicate functional, structural, or evolutionary relationships.

Global alignment

Global alignment aligns sequences from end to end.

Local alignment

Local alignment finds the most similar regions within sequences.

Gaps in alignments

Gaps are insertions or deletions introduced to optimize alignment. They affect the alignment score by introducing penalties.

Evolutionary interpretation of a good alignment

A good alignment suggests that the sequences share a common ancestor and have conserved regions due to functional or structural constraints.

Optimal alignment approaches

Optimal approaches, like dynamic programming, guarantee the best alignment but are computationally intensive.

Heuristic alignment approaches

Heuristic approaches, like BLAST, are faster but may not always find the optimal alignment.

BLAST

BLAST stands for Basic Local Alignment Search Tool. It is used to compare a query sequence against a database to find similar sequences.

BLAST search process

BLAST uses a sliding window approach to break the query into words, finds neighborhood words, extends alignments to form High-scoring Segment Pairs (HSPs), and ranks results based on scores.

Main fields on NCBI's BLAST tool page

Key fields include the query sequence, database selection, algorithm parameters, and optional filters for refining the search.

Steps BLAST takes to perform a search

Word Generation: Break the query into short words. Word Matching: Find matching words in the database. Extension: Extend matches to form HSPs. Scoring: Score and rank the HSPs. Output: Display the results with scores and alignments.

Major sections of a BLAST results page

Interpret the major sections of a BLAST results page.

Results Page

The results page includes the query sequence, database matches, alignment scores, E-values (indicating statistical significance), and detailed alignments showing mismatches and gaps.

Cladograms

Cladograms show relationships without indicating time.

Chronograms

Chronograms include time.

Phylograms

Phylograms show evolutionary distances.

Monophyletic Groups

Monophyletic groups include an ancestor and all its descendants.

Polyphyletic Groups

Polyphyletic groups include unrelated organisms.

Paraphyletic Groups

Paraphyletic groups include an ancestor and some, but not all, descendants.

Phylogenetic Trees as Hypotheses

Phylogenetic trees represent hypotheses about evolutionary relationships based on available data and can be tested and refined with new information.

Parts of a Phylogenetic Tree

Key parts include branches (representing evolutionary paths), nodes (common ancestors), and leaves (current species or sequences).

Evolutionary Relationships

The tree shows how species or sequences are related through common ancestors, with closely related species sharing more recent common ancestors.

PCR (Polymerase Chain Reaction)

PCR amplifies specific DNA sequences using cycles of denaturation (separating DNA strands), annealing (binding primers to target sequences), and extension (synthesizing new DNA strands). The result is a large quantity of the target DNA sequence.

Sanger Sequencing

Uses dideoxy nucleotides to terminate DNA synthesis, allowing sequence determination by fragment length.

Illumina Sequencing

Uses sequencing by synthesis with optical detection of incorporated nucleotides.

Ion Semiconductor Sequencing

Detects nucleotide incorporation by measuring changes in pH.

Nanopore Sequencing

Reads DNA sequences by detecting changes in electrical current as DNA passes through a nanopore.

PacBio Sequencing

Uses real-time sequencing by synthesis with long read lengths.

Dideoxy Nucleotides (ddNTPs)

Researchers use dideoxy nucleotides (ddNTPs) to terminate DNA synthesis at specific points.

Gel Electrophoresis

By running the resulting fragments through gel electrophoresis, they can determine the sequence based on fragment length.

Sequencing by Synthesis

Sequencing by synthesis involves synthesizing a complementary DNA strand and detecting the incorporation of nucleotides.

Genome Fragmentation

Technologies like Illumina and Ion Semiconductor fragment genomes before sequencing to create smaller, manageable pieces for analysis.

Short Reads

Illumina (100-300 bp), Ion Semiconductor (200-400 bp).

Long Reads

Nanopore (up to several kb), PacBio (up to 20 kb).

Genome Assembly Problems

Problems include repetitive sequences, gaps, and errors in read alignment, which can complicate the assembly process.

Reads

Short DNA sequences generated during sequencing.

Paired-End Reads

Sequences from both ends of a DNA fragment, providing more information for assembly.

Single-End Reads

Sequences from one end of a DNA fragment.

Contigs

Continuous sequences of DNA assembled from overlapping reads.

Scaffolds

Groups of contigs linked together using paired-end reads or other information.

De Novo Assembly

Assembling a genome without a reference, using only the sequence data.

Reference-Based Assembly

Aligning reads to a known reference genome to guide assembly.

Gene Expression Analysis

Involves measuring the amount of mRNA to infer gene activity. Techniques include Northern blots, qPCR, microarrays, and RNAseq.

Northern Blots

Detect specific RNA sequences using complementary probes.