Bio validation

gel electrophoresis, PCR, DNA sequencing

What is gel electrophoresis?

Where DNA is separated and anaylsed based on the charge, separating the fragments of DNA according to size on a gel.

What charge does DNA have?

A negative charge, allowing it to move through the gel as it has a negative charge on the phosphate group.

What does the electric current do?

It drives negatively charged DNA/RNA through a gel, separating the fragments according to size (smaller molecules move faster than larger)

What are the steps of gel electrophoresis?

1. Weighing out the agarose

2. Adding buffer to agarose

3. Melting the agarose solution

4. Pouring agarose into a mold - usually here DNA binding dye is added to the gel

5. Removing the set agarose gel (highly purified seaweed)

6. Gel is placed in a buffer-filled chamber

7. Loading DNA into the wells

8. Current is applied *positive and negative electrode

How are the bands visualised?

Under UV light, the DNA binding dye allows the DNA to be visible

The fragments appear as bands, separated according to their size.

There should be a DNA ladder present to compare the other bands to

Application of gel electrophoresis?

• Molecular biology

• Genetics

• Biochemistry research for DNA fingerprinting

• Gene expression analysis

• Protein purification

• Conservation: breeding programs

Why are breeding programs important?

They help conserve endangered animals

They aim to create the most genetic diversity possible - this means disease is less likely and greater resistance.

Inbreeding - aim away from, makes deletrious alleles, disease prone.

What does more amount of bands mean?

It shows how related the organism is to another, more bands = more chance of being related

What are restriction enzymes?

DNA cutting enzymes, where each one recognises one or a few target sequences, and cuts DNA at/near these sequences

How do restriction enzymes cut?

They make staggered cuts, producing ends with single-stranded DNA overhangs, or blunt ends.

It cuts then travels along the molecule, cutting the hydrogen bonds and then cutting the other strand - DON’T COUNT THE OVERHANG ONLY SECTIONS INBETWEEN

What are restriction fragments?

Molecular scissors that cut DNA into smaller pieces

What are restriction sites?

Restriction enzymes that only cut specific sequences of DNA

What is PCR?

Polymerase chain reaction

Replicates the wanted section of DNA enough times to be able to visualise the section in an agarose gel

Imitates natural DNA replication and amplifies DNA (Makes lots of copies)

What is the process of PCR?

Involves repeatedly heating and cooling a reaction mixture containing DNA template, primers, nucleotides, and heat-stable DNA polymerase (Taq) enzyme.

Each process typically involves 20-40 cycles and can generate millions/billions of copies of the target segment from the tiny amount of starting DNA

Steps of the PCR cycle:

Denaturation, annealing and extension

What is denaturation?

When the DNA sample is heated to 94-95 degrees to break the hydrogen bonds and separate DNA strands (into single)

What is annealing?

When the DNA sample is cooled to 50-60 degrees, to anneal/reattach primers to the DNA - telling polymerase where to start - this is for new strand synthesis

What is extension?

When the DNA sample is heated to 72 degrees (optimal temperature) to allow for Taq polymerase to add free nucleotides and extend strands - allows it to finish

What is needed for PCR?

• Buffer

• DNA template

• Forward and reverse primer

• Taq polymerase *found in bacteria

• Supply of nucleotides

• Thermocycler - able to rotate through necessary temperatures

• Cofactor

Application of PCR?

• DNA sequencing

• Disease

• Gene expression analysis

What is DNA profiling/fingerprinting?

Technique used by scientists to identify an individual by comparing an unknown sample of DNA with known profiles

What is a DNA profile?

A unique set of nucleotide fragments, displayed as a unique set of band proteins (by GE), that identifies individuals within a species

What is a STR

Short tandem repeat

Long stretches of DNA - usually sections of non-coding DNA repeated many times

Repeated sequence can be cut using restriction enzymes, amplified with PCR, and fluorescently tagged. Length can be determined using GE - due to mutation individuals will have different number of repeated sequences

Pair found on locusts of one pair of homologous chromosomes

Summary of the steps of DNA profiling?

1. Isolating the DNA sample from any somatic/body cell. A specific fragment is cut at a recognition site using restriction enzymes - amplified using PCR

2. PCR makes copies of the small amount of DNA

3. Fragments can be separated, length visualised, and number of repeats determined by GE. Smaller fragments have fewer STR’s and migrate further

4. DNA is visualised under UV light

5. Profile is the unique set of patterns of bands, which are different as we are all genetically diverse/unique

VNTR + STR:

Variable number tandem repeats

• Present in all members of population but number of repeats varies between individuals

• Banding pattern for offspring is a combination of both parents, but unique to the parents

  • Except for identical twins

Application of DNA profiling?

• Forensic investigations: linking suspects, etc.

• Paternity testing: determining biological relationships

• Conservation: identify and tracking endangered species

What is DNA sequencing?

Process of determining the order of nucleotides in a DNA molecule

All methods break down DNA into smaller fragments and read the sequence of nucleotides within in each fragment

Requirements for DNA sequencing

• Template DNA

• Short DNA ‘primer’ complimentary to DNA sequenced

• DNA polymerase

• 4 nucleotides and dideoxynucleotides

What is Sanger Sequencing ?

Most common method where DNA polymerase enzyme extends a primer along a single-stranded DNA template (like PCR)

4 separate reactions

Determined by analysing sequence of fragments from smallest to largest

Reliable and accurate method to sequence small to medium DNA fragments

What is next-generation sequencing (NGS)?

Sequencing many DNA fragments in parallel using high-throughput sequencing technologies

Rapid sequence of large amounts of DNA at a lower cost

Applications: genetic research, forensic analysis, and evolutionary biology

Scientific discoveries: sequence of human genome, identification of gene-causing mutations, and reconstruction of evolutionary relationships between species.

Summary of process of DNA sequencing

1. Mixture is heated and template strands separate

2. Temperature lowered, short primer finds its complimentary sequence. Temperature is raised slightly, allowing enzyme to bind to DNA and create a new strand

3. Sequencing reaction transferred from tube to lane of gel

4. Fragments migrate according to size

5. Each type of Dideoxynucleotides emit coloured light, and is recorded as a coloured band on the stimulated gel image

6. The coloured peaks represent each letter in the sequence

Example of DNA technologies for agriculture

Identification technologies have been used to accurately trace the genetics of desirable traits and to pass those traits to other plants within a generation

Using marker-assisted breeding, plant scientists can examine the DNA of seeds to find the ones that will produce the best plants

What is recombinant DNA technology?

Tools and techniques used to transfer a gene from a cell of a member of one species to the genome of a different species

What is bioengineering?

The combination of biology and engineering tools to create a usable product like a transgenic organism

What is biotechnology?

The use of living organisms and biological systems and processes for human benefit

Used in agriculture to improve yield, quality, and productivity of animals, however it reduces genetic diversity

What is a transgenic organism?

GMO’s (genetically modified organisms), that have been engineered for desirable traits, including:

• Disease resistance

• faster growth rate

• Greater product quality and yield

• Tolerance to adverse environmental conditions

Example of transgenic organisms in agriculture?

• Plants resistant to certain herbicides, allowing farmers to easily control the weeds in their fields

• Lupin varieties were developed to have increased levels of protein and reduced levels of alkaloids; more suitable for animal feed

• Wheat varieties resistant to certain pests and diseases, such as Russian wheat aphid

What is an example of a herbicide resistant crop

A crop genetically modified to withstand specific herbicides, allowing for effective weed control without damaging the crop itself.

“Round-up ready” - tolerant to herbicide containing glyphosate, preventing them from producing essential amino acids and causing them to die.

what was the first genetically modified animal?

Atlantic salmon - not approved in Australia

Its purpose was to increase the speed that the fish grows without affecting its ultimate size or other qualities

DNA technology in conservation?

• Maintaining genetic diversity is a priority for conservation

  • The more genetic diversity, the healthier the population and the higher chance of long term surivival

What are the ways that biotechnology can be used in environmental conservation for?

• Monitoring endangered species

• Quarantine

• Assessing gene pools for breeding programs

Expand on endangered species in DNA technologies

• Understanding the levels of genetic diversity may lead to genetic rescue

• DNA technologies to identify individuals using environmental DNA from hair, feces, etc.

• It is crucial as it helps scientists identify species threatened with extinction and provides evidence of effective conservation strategies

Breeding programs in DNA technologies

• Uses DNA profiling to identify individuals to avoid inbreeding depression (unmasking the deletrious alleles)

• Identify individuals that will maximise the genetic diversity

• Uses DNA fingerprinting to identify genetic diversity of restoration of rare plants

Quarantine in DNA technologies

• Identifies pest species through DNA fingerprinting

what does conservation planning to maintain viable gene pools include?

Consolidation of:

• Biogeography

• Population dynamics

• Reproductive behaviour

Biogeography in DNA technologies

• Identify genetic structure of a species across the species range (how large are the habitats needed)

• Understands historical species ranges to understand the results of climate change

population dynamics in DNA technologies

• The study of relatedness and breeding patterns in a species

• Uses DNA sequencing to identify new species

• E.g. Pollen dispersal patterns through paternity analysis

• Population growth and whether genes have come from outside of the population

• It is directly affected by the number of birth, deaths, immigration and emigration

What are the factors of population dynamics?

• Population growth: and size (key when planning conservation) - small populations have a higher risk of losing genetic diversity (smaller gene pool) - especially due to genetic drift

• Density: e.g. logging, or infectious disease - causing major changes. Habitat changing size and health - leading to limitations of food and resources (density dependent - causing increased competition and predation in the population)

• Migration: changes in the amount of individuals in the population

  • Emigration

  • Immigration

• Urbanisation

Reproductive behaviour in DNA technologies

• The behaviour related to the production and care of offspring, including establishment of mating systems, courtship, sexual behaviour, fertilisation and raising young

• Reproduction produces viable, fertile offspring that will also reproduce, perpetuating the species

• Reproductive behaviour must be considered when planning conservation strategies to prevent inbreeding (loss of advantageous alleles), gene pool diversity and reproductive fitness