Cell division
Two types of cell division: Mitosis and Meiosis
ü Mitosis: occurs in somatic cells and results in cell duplication→ two diploid daughter cells which are genetically identical both to each other and the original parent cell.
ü Meiosis: occurs in the germ cells of the gonads and is also known as ‘reduction division’ because it results in four haploid daughter cells, each containing just one member (homologue) of each chromosome pair, all genetically different.
Def: studying chromosomes in a form suitable for analysis during metaphase or Chromosomes are stained and visualized under a microscope.
Steps:
1. As chromosomes are only visible microscopically in dividing cells (metaphase chromosomes)
2. Chromosome analysis involves taking some non-dividing cells (usually peripheral blood lymphocytes)
3. Culturing them to encourage cell division on certain media.
4. Arrest of cellular division in metaphase.
5. Adding hypertonic saline leading to bursting of cells & release of chromosomes.
6. Staining which creates unique banding patterns on the chromosomes and subsequently examines the cells under microscopy for any extra or missing chromosomes, or large scale structural chromosomal abnormalities.
This method will miss any changes smaller than 5–10 megabases (5–10 million bases) in size, so is unsuitable for the detection of most microdeletions or duplications.
Along with array comparative genomic hybridization it is helpful in identifying changes in amount of genetic material – gene copy number variation (CNV) including duplications (increased copy number) and deletions (decreased copy number), so they are useful Investigate broad/complex phenotype.
Using Fluorescent-labelled DNA probes which are designed that are complementary to the DNA sequences being assessed, to detect and localize the presence or absence of specific DNA sequence on Chromosome.
Steps:
ü Chromosomes are immobilized and denatured on a microscope slide and exposed to a solution containing a fluorescently labelled probe specific to a specific chromosomal region.
ü After hybridization (the formation of a double strand of DNA from complementary single strands), the slide is washed and examined microscopically.
ü Where the probe has hybridized, fluorescent spots are seen over the relevant chromosome.
For example, if a child were suspected of having 22q11 deletion syndrome, FISH using a 22q11-specific probe would show only one pair of fluorescent spots, rather than two.
Types of FISH probes:
ü Centromeric (aneuploidy)
ü Telomeric (subtelomeric rearrangements)
ü Sequence specific (microdeletions)
ü Whole chromosome paint (complex rearrangements)
ü Reverse painting (to identify origin of unidentified chromosomal material)
Useful for microdeletion syndromes.
Ü Human genes: They are units of DNA sequences that containing information which determine the composition of an RNA molecule and most often translated to a protein.
Ü Every three nucleotides "triplet" represent a single codon, coding for a particular amino acid. Some codons act as a “start” signal, whereas others serve as “stop” signals that terminates translation .
Protein synthesis steps (From gene to protein)
1. DNA replication
ü Replication is semiconservative, i.e. each daughter molecule receives one strand from the parent DNA molecule.
ü Unwinding proteins, DNA-directed RNA polymerase, DNA polymerase and ligase are also required.
ü DNA polymerases synthesize the new strand in a 5' to 3' direction.
ü Discontinuous replication, i.e. one or both DNA strands may be synthesized in pieces known as Okazaki fragments, which are then linked together to yield a continuous DNA chain.
2. Transcription
ü Synthesis of complete RNA molecules from DNA by the enzyme RNA polymerase.
ü Transcription yields three types of RNA: mRNA, tRNA and rRNA.
ü Each nucleotide in the mRNA is complementary to one in the DNA template.
3. Post-transcriptional processing: the initial transcript is edited to remove introns and splice exons together by means of an RNA-protein complex called the spliceosome. Multiple different transcripts may be produced (alternative splicing).
Gene switch on/off
˗ Each cell expresses (turns on) only a fraction of its genes. The rest of the genes are repressed (turned off).
˗ The process of turning genes on and off is known as gene regulation.
˗ Gene regulation is an important part of normal development.
˗ Genes are turned on and off in different patterns during development to make a brain cell look and act different from a liver cell or a muscle cell, for example.
˗ Gene regulation can occur at any point during gene expression, but most commonly occurs at the level of transcription (when the information in a gene’s DNA is transferred to mRNA).
