DNA Replication mechanisms

• This chapter discusses basic genetics, focusing on blood bankers.

Classic Genetics

• Understanding the inheritance of blood group antigens and testing for disease markers are the most important areas of genetics.
• Mendel's First Law: Individual traits are inherited separately due to independent segregation.
• Mendel's Second Law: Genes for different traits are inherited separately due to independent assortment.
• Exception: Genes for separate traits can be inherited together as a single unit if closely linked on a chromosome.

Hardy-Weinberg Principle

• Mathematical formula to study Mendelian inheritance.
• p + q = 1, where p is the gene frequency of the dominant allele and q is the frequency of the recessive allele.
• p²+2pq+q² = 1;
• A three allele system equation is P + q + r = 1 or P²+2Pq+2Pr+ q² +2qr+r² = 1.

Inheritance Patterns

• Autosomal traits are those not carried on sex chromosomes.

Autosomal Recessive Inheritance

• A recessive trait is expressed only if both parents carry the trait, but it doesn't appear unless inherited from both.
• A dd genotype represents RhD negative, indicating that having one dominant gene is sufficient to express the trait.
• Dominant genes can be heterozygous or homozygous.

X-linked Dominant Trait

• The gene is present on the X chromosome, and inheriting one gene is enough to express the trait.

X-linked Recessive Trait

• Appears when the gene is homozygous in XY or X°X°, as seen in hemophilia A (where X° represents the affected gene).

Autosomal Dominant

• Only one gene is needed to express the trait

Cellular Genetics

Mitosis

• Cell division resulting in two identical cells with the same number of chromosomes as the mother cell.
• Mitosis complex process is divided into five stages: Interphase (2N), Prophase (4N), Metaphase, Anaphase (4N), and Telophase (2N).

Meiosis

• Process to produce gametes.
• One cell (2N) divides into 4 unique cells (1N).

Stages of Mitosis

• Interphase (2N): Resting stage where cells synthesize RNA and proteins, and chromatin is uncondensed.
• Prophase (4N): Chromosomes become visible and condense, with each chromosome having two chromatids linked by a centromere.
• Metaphase (4N): Chromosomes move to the cell's equator, held by microtubules attached to the mitotic spindle apparatus.
• Anaphase (4N): Sister chromatids separate and move towards opposite poles, with a decrease of cell diameter at the equator.
• Telophase (2N): Chromosomes reach the poles, and the cell membrane divides, resulting in two cells each containing a set of chromosomes identical to the parent cell.

Stages of Meiosis

• Interphase (2N): Resting stage where cells synthesize RNA and proteins, and chromatin is uncondensed.
• Prophase I (4N): Chromosomes condense, homologous chromosomes pair to form bivalents, and crossing over occurs.
• Metaphase I (4N): Bivalent chromosomes align at the cell equator, each containing all four copies of each chromosome.
• Anaphase I (4N): Homologous pairs move to opposite poles, with sister chromatids remaining together.
• Telophase I (2N): The cell divides into two daughter cells, each with 2N chromosomes.
• Metaphase II (2N): Homologues line up at the equator.
• Anaphase II (N): Homologues move to opposite poles of the equator.
• Telophase II (N): Each cell separates into two new cells, resulting in four cells with unique genetic constitutions.

Cell Division Stages

• Go (Cap-0): "Resting stage" with no cell division.
• G1: "Pre-replication stage" (Gap-1) where cells produce RNA and synthesize protein (2N).
• Synthesis: DNA replication occurs.
• G2: "Post-replication stage" (Cap-2) where the cell synthesizes RNA and protein (4N).
• M phase: Mitosis (cell division) occurs (2N).

Molecular Genetics

• DNA comprises four nitrogenous bases, a sugar molecule called deoxyribose, and a phosphate group.
• The four base letters are Adenine (A), Cytosine (C), Guanine (G), and Thymine (T).
• Phosphates attach to the sugar at the third and fifth carbon atoms that forms backbone.
• DNA strands are antiparallel, with one strand is 5' to 3' and the complementary strand 3' to 5'.
• Triplets of nucleotides called codons code for specific amino acids.

Replication

• Process by which DNA is copied before mitosis and cell division.

Steps of Replication

• DNA gyrase undoes supercoils.
• DNA helicase separates the two strands of duplex DNA.
• DNA polymerase III synthesizes a new strand in the 5'-3' direction on the leading strand.
• DNA polymerase III proofreads the addition of new bases, removing incorrectly incorporated ones.
• Single-stranded binding proteins interact with opened DNA strands to prevent hydrogen bonding.
• DNA polymerase I and DNA Ligase join Okazaki fragments on the lagging strand
• Primase synthesizes RNA and adds it to DNA.
• DNA Ligase joins the phosphodiester bonds of the DNA backbone.
• Isomerase enzyme recoils the DNA.

Repair

• DNA must be copied exactly to maintain vitality.
• Proof reading ability of DNA polymerase.
• Editing strand by mismatch repair.

DNA Repair System

• Photo reactivation (PR) enzyme.
• Excision repair.
• Recombinational repair.
• Mismatch repair.
• SOS repair.

Factors that alter DNA by modifying it chemically or physically

• Alkylating agents react with guanine, causing depurination.
• Ionizing radiation (peroxides) causes single-strand breaks.
• Mitomycin C (strong oxidant) forms covalent linkages between bases on opposite strands.
• Ultraviolet radiation alters thymine bases, forming thymine dimers.

Mutation

• Any change in the DNA sequence structure, whether physical or biochemical.
• Mutations can be spontaneous.
• An organism with a mutation is a mutant, with the original sequence as the wild type.
• Mutagens are any chemical or condition that causes mutation.

Type of Mutation

Point Mutation

• A change in a single nucleotide in the DNA which includes the below.

Substitution

• Replacing one nucleotide with another.
• Transition: Replacing a purine with another purine (A ↔ G) or a pyrimidine with another pyrimidine (T ↔ C).
• Transversion: Replacing a purine with a pyrimidine or vice versa.

Insertion

• Addition of a new nucleotide.

Deletion

• Removal of a nucleotide.
• Transition is when one purine/pyrimidine is substituted for another.

Transversion

• Where a purine is substituted for a pyrimidine or vice versa.

Silent Mutation

• A change in the DNA sequence does not alters the amino acid sequence due to redundancy in the genetic code and that part not seem critical for its function.
• Threonine has multiple codons (ACA, ACC, ACG, ACU), changes the third nucleotide which does not affect the resulting amino acid.

Missense Point Mutation

• Change results in a different amino acid in the corresponding peptide, affects protein function and can result in hemoglobin disorders.
• Mutations in hemoglobin cause different types of inherited anemias.

Nonsense Mutation

• A point change leads to the formation of a stop codon (Amber, Opal, Ochre), resulting in a truncated peptide.

Mutation Impact

• A nucleotide change resulting in a stop codon (UAG, UGA, UAA), which leads to a truncated protein and loss of function is a key mutation feature.

Insertion or Deletion

• Impact is significant when one or two nucleotides are involved, causing a frameshift if not in multiples of three.

Gross Mutation

• Duplication, recombination, and large deletion have lower frequency than replication mutations.

Deletion of Large Segment of DNA

• Mutagens are chemical substances or environmental conditions causing mutations and are often carcinogens, giving affected cells a growth advantage.
• Mutations can be harmful, neutral, or beneficial and multiple repair systems highlight the importance of maintaining DNA integrity.

Molecular Genetics: RNA

• RNA is single-stranded, contains uracil instead of thymine, and has a ribose sugar.
• RNA is translated into peptides and proteins in ribosomes, and transcription copies DNA to RNA.

Type of RNA

• Messenger RNA
• Ribosomal RNA (rRNA) makes up much of the ribosomal structure on the endoplasmic reticulum.
• RNA polymerase I transcribes rRNA in the ribosome, where protein synthesis that takes place.
• Messenger RNA (mRNA) is a linear sequence transcribed from DNA that carries information to ribosomes for protein synthesis and RNA polymerase II transcribes mRNA.
• Transfer RNA (tRNA) transfers amino acids to the ribosome and RNA splicing removes introns and remains exons on mRNA "mature".

Transcription

• Cellular process of copying DNA to RNA, synthesizing RNA in the 5' to 3' direction.
• MRNA is protected by a 7-methyl guanosyl cap at 5', mature mRNA is transported out of the nucleus.

Translation

• The process by which RNA transcripts are turned into proteins and peptides.

Translation Stages

• Initiation is the attachment of free Methionine to a transfer RNA molecule (tRNA).
• Elongation is next.
• Termination occurs when the ribosome encounters a stop codon (UAA, UGA, UAG) and separate mRNA from ribosome.

Modern Genetics Techniques

• Isolate material with minimum structure damage.
• Must be away to visualize and locate the molecular species.
• Method to separate out different species.
• Method to quantify the isolated and studied specifies has to be used to get different exact as possible in the process studied.

DNA Isolation

• Involves alkaline denaturation and precipitation with alcohol.

Process details

• Nucleic acids are purified using specific columns that bind based on charge interaction, has greater care required than DNA. Complimentary DNA (cDNA) synthesis converts RNA to DNA in vitro. Once isolated, DNA can be store in low salt buffers @-20°C on RNA @-80°C.

Sequence Determination involves

• Direct sequencing by chemically cleaving DNA based on nucleotide presence.
• Termination using DNA polymerase.

Electrophoresis

• Comparison of DNA and RNA is done to a process called gel electrophoresis.
• Cloning makes an exact copy of a desired DNA sequence.
• PCR amplifies isolated pieces of DNA or RNA using DNA polymerase and heating/cooling.

Important Points

• Genetics studies inheritance or transmission of characteristics from parents to offspring, based on the biochemical structure of chromatin.
• Chromatin includes nucleic acids, structural proteins, and enzymes for genetic processes like replication.
• All organisms have specific numbers of chromosomes: humans have 22 pairs of autosomes plus one sex chromosome pair (XX for females, XY for males), totaling 46 chromosomes.
• Mendel's law says factors for different characteristics are inherited independently if they reside on different chromosomes.
• Human chromosomes are chromatin, which is DNA wrapped around histones, and DNA's helical structure allows much information to be packaged in a small space.
• DNA replication is semi conservative and uses DNA polymerase to produce a complementary strand, with each strand acting as a template. DNA is always read and written in the 5' to 3' direction.
• Mutation is any structural alteration of DNA caused by a physical or chemical agent, and mutations can be beneficial or deleterious. Some are lethal, while others are silent.
• Transcription is an enzymatic process where a DNA strand is copied into mRNA. Eukaryotic mRNA is altered via processing steps like intron removal and poly-A tail addition before being exported for translation.
• Translation is the complex process of turning mRNA into proteins, which are the functional units of an organism. It occurs on ribosomes, and proteins require specific forms like disulfide linkages.
• Varied methods manipulate DNA, forming genetic engineering, Southern blotting, sequencing, and cloning analyze DNA, Northern blotting RNA protection assays analyze RNA. Western blotting and immunoprecipitation are used for proteins.
• Restriction endonucleases found in bacteria cut DNA at specific sites, with digests having unique patterns.
• A DNA probe is a piece of DNA with a sequence for a specific gene, labeled and visualized by autoradiography or fluorescent imaging.
• Polymerase chain reaction (PCR) is synthesized by DNA sequences using nucleotide primers that hybridize to DNA strands.
• A cloning vector is an extra-chromosomal element that carries recombinant DNA into a host cell. Most vectors resistant for antibiotic markers or fluorescent proteins
• Sources of DNA for analysis include prokaryotic or eukaryotic cells as well as forensics. Active cells provide good RNA.
• There are many new techniques in use that help to understand the complete nature of genetic material and how it functions.