Module 2: Molecular Biology Techniques

How do we analyze DNA?

• PCR method (polymerase chain reaction)

• used for DNA amplification and cloning

What is DNA analysis?

• involves multiple techniques to study the structure, sequence and function of DNA

How does PCR work?

• molecular biology technique for DNA cloning

• you need to know part of the gene’s DNA sequence to create primers

• primers: short pieces of synthetic DNA sequences that matches the target gene

• these primers help make several copies of the gene, which then can be used in cloning or other lab techniques

Primers

• short pieces of synthetic DNA sequences that matches the gene that you are targeting

• these primers help make multiple copies of the gene, which can be used in cloning

How are restriction fragments used in recombinant DNA technology?

1. Cutting DNA with a Restriction Enzyme

   • DNA from 2 different sources is treated with a restriction enzyme

   • the enzyme cuts the DNA at specific sequences

   • this creates fragments with sticky ends (single-stranded overhangs)

2. Mixing Fragments for Base-Pairing

   • the sticky ends can base-pair with complementary sequences

   • it can form hydrogen bonds with sticky ends from another DNA molecule

3. Sealing with DNA Ligase

   • DNA ligase (enzyme) seals the backbone of the DNA fragments

   • this forms a stable recombinant DNA molecule

Restriction Enzymes

• enzymes that cut DNA at specific sequences called restriction sites

• restriction sites are 4-8 base pairs long

• sites are often palindromes - they read the same forward and backward on opposite strands

• this allows for the enzyme to cut both strands at the same spot

Palindromes

• where it reads the same forward and backward on opposite strands of a restriction site

• allows for the enzyme to cut both strands at the same spot

Restriction Sites

• a specific short sequence which is 4-8 base pairs long

• site of DNA cutting involving restriction enzymes

2 types of DNA cuts made by restriction enzymes

1. Blunt Ends

   • cuts straight across both strands

   • no overhangs

   • harder to join, but useful in some cloning applications

2. Sticky Ends

   • cuts at staggered positions

   • resulting in single-stranded overhangs

   • these overhangs can easily base-pair with matching ends

   • easier to join for cloning

Recombinant DNA Technology

• allows scientists to combine DNA from different sources to create new DNA molecules

• instead of changing proteins directly, scientists change the DNA that codes for them

• DNA → RNA → Protein (central dogma)

• so, changing DNA lets you control which proteins are made

DNA Cloning

• a molecular biology technique that involves making copies of a specific DNA sequence

DNA Transformation

• molecular biology technique

• adds foreign DNA into a cell to express new genetic material

• transforms that into bacteria, yeast or plant cells

Gel Electrophoresis

• molecular biology technique that separates DNA fragments based on their size

How does Gel Electrophoresis work?

1. DNA fragments of different lengths is loaded into the wells in a gel matrix

2. An electric current is applied, causing the fragments to migrate

3. Since DNA is negatively charged, it moves toward the positive electrode (anode)

4. Smaller fragments move faster and farther through the gel, compared to the larger ones

Visualizing DNA:

• DNA is stained with EtBr (ethidium bromide)

• Under UV light, EtBr makes the DNA glow orange

• The bands to become visible, representing different fragment sizes

During electrophoresis, what will be the behaviour of smaller and larger DNA fragments on the gel?

1. Smaller DNA fragments will run faster while larger DNA fragments will run slower

2. Smaller DNA fragments will run slower while larger DNA fragments will run faster

3. They will run the same time

Smaller DNA fragments will run faster while larger DNA fragments will run slower

How do we analyze genomes?

• whole genome sequencing

• allows scientists to decode and read the entire genome of an organism

• this technology was used 25 years ago with human genome

• reveals information such as gene functions, mutations, and inheritance patterns

3 types of genomes in eukaryotic cells

1. Nuclear Genome

   • found in the nucleus

   • contains most of the organism’s DNA, stored in chromosomes

2. Mitochondrial Genome

   • small, circular DNA, found in the mitochondria

   • only inherited maternally

3. Chloroplast Genome

   • found in chloroplasts (plants only)

   • carries genes that are important for photosynthesis

Pyrosequencing

• type of next-generation DNA sequencing technology

• detects DNA bases (A,T,C,G) as they’re added in real time

• uses bioluminescent reaction (light-producing) to show when a base is added

How does Pyrosequencing work?

1. Ingredients for Reaction Mix

   • 4 labelled bases (A,T,C,G)

   • Luciferase (enzyme that makes light)

   • DNA polymerase (builds DNA)

   • ATP sulfurylase, luciferin (necessary enzymes)

2. Adding a Nucleotide

   • the correct nucleotide is added to the growing DNA strand during sequencing

   • the releases PPi (pyrophosphate) as a byproduct

3. Light Signal

   • PPi is converted into ATP by ATP sulfurylase

   • enzyme Luciferase uses this ATP to produce light

   • a sensor detects the light flashes, telling scientists which base was added

   • each light flash = one nucleotide added = DNA sequence being revealed

illumina Sequencing

• type of next-generation DNA sequencing technology

• it uses Sequencing by Synthesis (SBS) technique to read DNA sequences in real time

How does illumina Sequencing work?

1. Fluorescently Tagged Nucleotides

   • each base (A,T,C,G) is tagged a unique fluorescent dye

   • these are added to the growing DNA strand during sequencing

2. Reversible Terminator

   • each base contains a reversible terminator

   • it temporarily blocks further base addition

   • it ensures that only one nucleotide is added at a time

3. Nucleotide Addition & Fluorescent Detection

   • a single nucleotide is added into the strand

   • a high-resolution camera detects the fluorescent tag to identify which base was added

4. Terminator Removal & Cycle Repeats

   • the reversible terminator is removed

   • the cycle is repeated for each base, building the full DNA sequence

2 types of next-generation DNA sequencing technology (NGS)

1. Pyrosequencing

2. illumina Sequencing

Next Generation vs Third Generation Sequencing

Next Generation DNA Sequencing:

• produces shorter read lengths of DNA

• analyzes larger amounts of DNA (high-throughput)

• requires DNA amplification (making multiple copies) before sequencing

• example: pyrosequencing and illumina sequencing

Third Generation DNA Sequencing:

• also known as long road technology

• produces longer read lengths of DNA

• it can read single DNA molecules directly (no DNA amplification)

• favours the study of complex genomic regions

• example: nanopore sequencing

Nanopore Sequencing

• a type of TGS (third-generation sequencing technology)

• produces longer reads of DNA

• does not require DNA synthesis

• DNA passes through a nanopore (protein-based pore) in a membrane

• reads DNA sequences in real time

How does Nanopore Sequencing work?

1. DNA Movement

   • a motor protein guides the single-stranded DNA through the nanopore

2. Electrical Signal Disruption

   • an electrical current is applied across the membrane

   • as the DNA moves through, each base causes a unique disruption in the current

3. Base Detection

   • the electrical signal changes are recorded

   • each base (A,T,C,G) has a distinct signal

   • this allows for the DNA sequence to be read in real time

Bioinformatics

• interdisciplinary field that combines biology, computer science and data analysis

• helps store, analyze and interpret biological sequencing data

Exome

• the part of the genome that code for proteins (exons only)

Transcriptome

• all RNA molecules made from a genome

Proteome

• all proteins produced in an organism

• more complex than the genome due to alternative splicing, post-translational modifications and protein interactions

Genetic Variation in Humans

• all humans are 99.7% genetically identical

• the remaining 0.3% (bases) makes us unique individuals (including traits, appearance and disease risk)

Genomics and Human Diseases

• genomic research helps us understand how mutations cause disease

• some diseases are caused monogenic (caused by single gene)

• others are multigenic (involve multiple genes + environmental factors)

What is the field of study dealing with all the proteins within a cell or organism? 

1. Genomics

2. Bioinformatics 

3. Proteomics

4. Exomics

5. transcriptomics

Proteomics

2 human diseases that cause genetic mutations

1. Sickle Cell Anemia

   • caused by point mutation in the β-globin gene (HBB)

2. Cystic Fibrosis

   • caused by mutations in a single gene (CFTR)

• Most diseases (diabetes, cancer) are multigenic