DNA Sequencing and BLAST Overview

Key Concepts of DNA Sequencing and BLAST Analysis

• Score (bits)

  • The numerical scores assigned by BLAST that indicates the quality of the sequence alignment. Higher bit scores indicate better alignment with the query sequence.

• E-value

  • An E-value of 3 or less represents an acceptable match, suggesting the alignment is statistically significant and not likely due to random chance.

• Scientific Name of the Bacterium

  • The bacterium sequenced is Bartonella henselae, which is pathogenic to humans and is transmitted via a vector or directly from an animal reservoir.

DNA Sequencing Techniques

• Purpose of Laser Beam

  • The laser beams are used to read the sequence of nucleotides based on their fluorescence as DNA fragments pass through the beam, enabling the determination of DNA sequences.

• Goal of Sequence Matching Analysis

  • The goal is to determine whether a new DNA sequence shares significant similarity (or homology) with known sequences in databases.

• Definition of Homology

  • Homology refers to the significant similarities observed between new sequences and existing sequences, which indicate evolutionary relationships.

• What is BLAST?

  • BLAST (Basic Local Alignment Search Tool) is a program that provides a combination of speed, sensitivity, flexibility, and statistical rigor to search nucleotide or protein databases for sequence similarities.

• Assumption in Evolutionary Relationships

  • A key assumption is that the number of differing positions in nucleotide sequences correlates with the elapsed time since the two species diverged from a common ancestor.

PCR and Gel Electrophoresis

• Sequencing Brew Components

  • The sequencing brew contains buffers, primers (specific to each tube), DNA polymerases, nucleotides, and fluorescence-tagged terminators, all in suitable proportions for sequencing reactions.

• Purpose of Second PCR

  • Unlike the first PCR, the second PCR aims to produce multiple copies of variable lengths of the desired DNA sequence, preparing it for sequencing.

• Source of Primers

  • Scientists obtain primers from commercial sources for use in PCR and sequencing techniques.

• Final PCR Product

  • The final product consists of a mixture of DNA fragments of varying lengths, contained in the PCR tubes, ready for further analysis.

• Purpose of Gel Electrophoresis

  • Gel electrophoresis separates DNA fragments based on size differences, allowing visualization and confirmation of successful PCR amplification.

• Movement of DNA in Relation to Charge

  • DNA molecules move towards the positive charge applied during electrophoresis because DNA has a negative charge due to its phosphate backbone.

PCR Cycle Details

• Electrophoresis Gel Use

  • Running an electrophoresis gel at this stage confirms success in the PCR reaction by visualizing DNA fragment sizes.

• Lane Contents in Gel

  • Negative Control: Contains only water, no DNA.

  • Positive Control: Contains known PCR product, confirming the PCR process.

  • Sample Lane: Contains the sample PCR products being tested.

• Cycle Sequencing Steps

  1. Use a thermocycler to create multiple copies of the target DNA, terminating replication at random places to generate varying lengths of sequences.

• Highly Variable Regions

  • Highly variable regions in DNA are crucial for identification purposes, as these regions provide distinct genetic markers among different species.

PCR Cycle Steps

• Each Cycle Steps (Temperature and Duration):
  a. Melt: 95°C for 30 seconds - DNA strands separate.
  b. Anneal: 60°C for 30 seconds - Primers bind to single-stranded DNA.
  c. Extend: 72°C for 45 seconds - DNA polymerase extends the DNA strand.

• Total Copies After Cycles

  • After 8 cycles, a total of 240 copies of the target DNA are synthesized, magnifying the initial amount significantly.