Molecular Biology Techniques

Microinjection

• Ensures entry and expression into target cells
• Requires technical expertise and equipment
• Only relatively few cells can be used

Viral Transfection

• Laboratory technique to introduce genetic material into cells using viruses as carriers
• Process involves the use of viral vectors
• Does not cause disease

'Bla' Gene

• Bacteria with this gene grow in the presence of ampicillin

GFP Gene

• Bacteria with this gene glow under near UV light

Automated DNA Sequencing

• Laboratory technique to determine DNA nucleotide order
• Primers attach to the template strand and DNA polymerase reads the template strand
• Synthesizes a new complementary second strand to match
• Strand will terminate synthesis when a “Terminator Base” attaches at the 3’ end
• Terminator bases have dideoxyTTP (ddTTP), which lack 3'-OH group, stopping DNA strand elongation
• Separate strands by Gel electrophoresis
• Each terminator base makes up the sequence of DNA, reading from the bottom (5’ to 3’)
• Identifies the terminal nucleotide in each fragment

Plasmid Transfer and Drug Resistance

• Plasmid is transferred from cell to cell, carrying a drug resistance gene
• Plasmid replication allows the bacteria to grow and replicate on the plate
• As the bacteria grow, they replicate not only themselves but also the plasmid with gene
• Population increases
• Selective screening identifies colonies that likely contain the target gene

LacZ Selection System

• The LacZ gene metabolizes X-gal, producing a blue product
• Insertion of foreign DNA into the MCS part of the LacZ gene destroys the LacZ gene, resulting in no blue product of X-gal formed in colonies (white)

Promoter Regions

• Determines the rate at which mRNA is synthesized
• Amount of protein expressed depends on the choice of promoter

DNA Preparation and Storage

• Two ways to prepare and store DNA:
  • Deactivate DNA damaging enzymes by heat/removal of divalent cations cofactors by chelating agents (e.g. EDTA)
  • Storage in ethanol at low temperatures
• Five popular sources of DNA:
  • Blood samples (must remain unclotted) collected in EDTA tubes
  • Amniocytes (from amniocentesis fluid)
  • Tissue biopsies
  • Cultured cells
  • Paraffin-embedded tissue samples (mainly tumours)

Cell Collection and Lysis

• Cells are normally collected using a water mouth wash to dislodge epithelial cells lining the mouth
• Transfer the sample into a microcentrifuge tube and centrifuge to extract cells
• During cell lysis, a lysis buffer is used, which consists of various components

Brain Structure

• The brain has four lobes: frontal, parietal, temporal, and occipital.
• The central sulcus divides the frontal and parietal lobes.
• The lateral sulcus (also called the Sylvian fissure) separates the temporal lobe from the frontal and parietal lobes.
• A gyrus is a brain crest, and a sulcus is a depression or dip.

Functions of Brain Lobes

• Frontal lobe: short-term memory, motor control, higher cognition, personality, foresight, and hindsight.
• Parietal lobe: processing of tactile and proprioceptive information, comprehension of language, and orientation of individual in space and time.
• Temporal lobe: auditory, emotional and visceral responses, learning and memory recall, and other functions.
• Occipital lobe: visual function.

Neurons and Glial Cells

• There are three types of neurons: sensory (afferent), relay (interneuron), and motor (efferent).
• Unipolar neurons have a single axon, bipolar neurons have two axons, and multipolar neurons have multiple axons.
• Pseudounipolar neurons have a single axon that divides into two branches.
• Glial cells are neural supporting cells that include Schwann cells, microglial cells, ependyma, astrocyte, and oligodendrocyte.

Functions of Glial Cells

• Schwann cells form the myelin sheath in the peripheral nervous system.
• Astrocyte functions: clearing excess neurotransmitters, regulating the blood-brain barrier, and promoting synapse formation.
• Microglial cells act like macrophages, clearing cellular debris and dead neurons from nervous tissue through phagocytosis.
• Ependyma cells control the production and flow of cerebrospinal fluid, brain metabolism, and waste clearance.
• Oligodendrocyte cells support myelinating cells of the central nervous system.

Nervous System Components

• The central nervous system (CNS) has 12 pairs of cranial nerves.
• The autonomic nervous system has two components: sympathetic and parasympathetic.
• The somatic motor system includes sensory neurons and motor neurons.

Spinal Reflex Arc

• The spinal reflex arc is an involuntary response to a stimulus.
• The arc involves a sensory neuron, an interneuron, and a motor neuron.
• The response bypasses the brain, making it faster.

Autonomic Nervous System

• The parasympathetic division is responsible for "rest and digest" functions.
• The parasympathetic division arises from the sacral spinal cord and has various effects on the body, including decreasing heart rate and increasing secretion of tears and saliva.
• The sympathetic division is responsible for "fight or flight" responses.
• The sympathetic division arises from the thoracic and lumbar spinal cord and has various effects on the body, including dilating the pupil, increasing heart rate, and constricting blood vessels.

Neuro-Endocrine System

• The neuro-endocrine system involves cell bodies in the nucleus of the hypothalamus releasing hormones into the blood by acting on glands.

The Nervous System

• The CNS has 12 pairs of cranial nerves.
• The Autonomic Nervous System has two components: Sympathetic and Parasympathetic.

Neuron Structure

• Sensory neurons are afferent.
• Relay neurons are interneurons.
• Motor neurons are efferent.
• Unipolar neuron structure is sensory and has an example.
• Bipolar neuron structure has an example.
• Pseudo-unipolar neuron structure has a specific function.
• Multipolar neuron structure is the most common type.

Glial Cells

• Glial cells are neural supporting cells.
• Examples of glial cells include:
  • Schwann cells
  • Microglial cells
  • Ependyma
  • Astrocyte
  • Oligodendrocyte
• Astrocyte functions include:
  • Clearing excess neurotransmitters
  • Regulating the blood-brain barrier
  • Promoting synapse formation
• Microglial cells function as macrophages, clearing cellular debris and dead neurons.

Brain Structure

• A gyrus is a brain crest, and a sulcus is a depression.
• The four lobes of the brain are:
  • Frontal lobe
  • Parietal lobe
  • Temporal lobe
  • Occipital lobe
• The central sulcus divides the frontal and parietal lobes.
• The lateral sulcus separates the temporal lobe from the frontal and parietal lobes.

Lobe Functions

• Frontal lobe functions include:
  • Short-term memory
  • Motor control
  • Higher cognition
  • Personality
  • Foresight and hindsight
• Parietal lobe functions include:
  • Processing tactile and proprioceptive information
  • Comprehension of language
  • Orientation of individual in space and time
• Temporal lobe functions include:
  • Auditory processing
  • Emotional and visceral responses
  • Learning and memory recall
  • Seeking and capturing prey
  • Courtship, mating, and rearing of young
  • Balance between aggressive and communal behavior
  • Formation of memory
  • Homeostasis
• Occipital lobe function is:
  • Visual function

Developmental Disorders

• Anencephaly is a serious birth defect in which a baby is born without parts of the brain and skull.
• Spinal Bifida is a type of neural tube defect, causing a gap in the spine.

Reflexes

• Approach reflexes are present at birth and include:
  • Breathing
  • Rooting
  • Sucking
  • Swallowing
• Avoidance reflexes include:
  • Coughing
  • Sneezing
  • Blinking
• Palma grasp is a primitive and involuntary reflex found in infants of humans and most primates.