Chapter 2: Basic Genetics for Blood Bankers - DNA, RNA & Mutation | PDF

**CHAPTER 2** 1. **Classic Genetics** - Most important area of the science of genetic to understanding of the inheritance of blood group antigen and the testing for disease markers at the molecular level. a. **Mendel's First Law** inherited separately from each other. b. **Mendel's...

**CHAPTER 2** 1. **Classic Genetics** - Most important area of the science of genetic to understanding of the inheritance of blood group antigen and the testing for disease markers at the molecular level. a. **Mendel's First Law** inherited separately from each other. b. **Mendel's Second Law** c. **Exceptions to the Mendelian Law of inheritance.** 2. **Hardy -- Weinberg Principle** 1. P + q = 1; p q --- ------ ------ p p^2^ pq q pq q^2^ A three allele system use P +q + r = 1 or P²+2Pq+2Pr+ q^2^ +2qr+r² = 1 3. **Inheritance Patterns** - Autosomal : refers to trait that are not carried on the sex chromosomes. A. Autosomal recessive inheritance:- A recessive trait carried by either parent or both parent but trait not appear unless both parents carry the trait. Like dd genotype represents RhD negative "one dominant gene inherited is enough to represent trait". Heterozygous or homozygous for dominant-gene. B. X-linked dominant trait gene present on X chromosome one gene inherited for trait enough to represent trait. C. X-linked Recessive. D. Autosomal-dominant - One gene is enough to represent trait. - **3. Cellular Genetics** 1. **Mitosis** The complex process of (original) mitosis divided into 5 stages: 2. **Meiosis** - Process to produce the gamete. - One cell (2N) divided to 4 unique cells (1N) Stage Box 2-1 box2-3.jpg 3. **Cell Division** The cell cycle is divided into four distinct stages: a. G~0~ Cap-0 "resting stage" no cell division. b. G1 "Pre-replication stage" Gap-1, cell produce RNA and synthesize protein (2N). c. Synthesis --DNA replication occurs. d. G2 "Cap-2" post-replication stage". During the gap between DNA synthesis and mitosis, the cell continues to synthesize RNA and produce a new protein (4N). e. M phase Mitosis-cell division occur (2N)  figure2-7.jpg 4. **Molecular Genetics** DNA is composed of four nitrogenous bases. A sugar molecule called deoxyribose of phosphate group. The four different bases are Adenine (A), Cytosine (C), Guanine (G) and Thymine (T). Phosphates in the DNA back bone attached to the sugar at the third and fifth carbon atoms. The two DNA strands are anti parallel that is one strand is 5' "prime" to 3' and the complementary strand 5' to 3' in other direction. Triplets of nucleotides called codon which response to code for one specific amino acid. 5. **Replication** Is the complex process by which DNA is copied before mitosis can occur and cell division. 6. **Enzyme and steps of replication:** - DNA gyrase → undoes the supercoils. - DNA helicase → separates the two strands of duplex DNA. - DNA polymerase III can synthesize a new strand in 5'-3' direction on the leading strand. - DNA polymerase III proof reads the addition of new bases to the growing DNA strands and can remove an incorrectly incorporated base. - Single-stranded binding proteins → interact with the opened strands of DNA to prevent hydrogen bonding. - DNA polymerase I and DNA Ligase joined the Okazaki fragment on the logging strand. - Primase synthesis of RNA and added to DNA. - DNA Ligase joins the phosphodiester bonds of the DNA backbone to complete the intact molecule. - Isomerase enzyme recoil the DNA. 7. **Repair** DNA must be copied exactly or the information will be altered which may lead to decrease in vitality of the organism. The mechanisms can be detected the mistakes and corrects the actual DNA sequence:- 1. 2. 3. a. b. c. d. e. Many common chemical and environmental factors can alter DNA by modifying it chemically or physically:- 1. 2. 3. 4. 8. **Mutation:-** 1. Mutation is any change in the structure of sequence of DNA, whether it is physical or biochemical. 2. None of the systems are fool proof and mutation occurs. 3. Mutagens is any chemical or condition can cause mutation. 4. Mutation can be spontaneous 5. An organism carrying a mutation is called a mutant, while the original DNA sequence is referred to as the wild type. **Type of mutation:-** 1. **Point mutation:- A change in a single nucleotide in the DNA sequence, Includes:** a. Substitution: Replacing one nucleotide with another. i. **Transition**: Substitution of a purine for another purine (A ↔ G) or a pyrimidine for another pyrimidine (T ↔ C). ii. **Transversion:** Substitution of a purine for a pyrimidine or vice versa. b. Insertion: Addition of a new nucleotide. c. Deletions: Removal of a nucleotide. **Transition** is type of mutation in which one purine pyrimidine substituted for another purine-pyrimidine like: **Transversion** **Transversion** is type of mutation in purine is substituted for pyrimidine or pyrimidine for purine like: 2. **Silent mutation** **Due to substitution one codon and which represent some amino acid or due to mutation in peptide sequence, but that part not seem critical for its function.** - **A change in the DNA sequence that does not alter the amino acid sequence in the resulting protein due to redundancy in the genetic code.** - **Example: Threonine has multiple codons (ACA, ACC, ACG, ACU), so changing the third nucleotide does not affect the resulting amino acid.** 3. **Missense Point Mutation** - A change in a nucleotide that leads to a change in the amino acid in the resulting protein. - May affect protein function. - Example: Mutations in hemoglobin causing different types of inherited anemias. 4. **Nonsense Mutation** - A change in a nucleotide that results in the formation of a stop codon (e.g., UAG, UGA, UAA). - Leads to a truncated protein, which may lose its function. 5. **Insertion or deletion** of one or two nucleotides in the DNA sequence which lead to large change in the amino acid sequence occurs like subgroup of A antigen - Addition or removal of nucleotides from the DNA sequence. - If not in multiples of three, it can cause a frameshift, completely altering the amino acid sequence. 6. **Gross Mutation** 7. **Deletion of Large Segment of DNA** **Mutagens:** - **Chemical substances or environmental conditions that cause mutations.** - **Many mutagens are also carcinogens because they give affected cells a growth advantage.** **Importance of Mutations:** - **Mutations can be harmful, neutral, or beneficial, depending on their effect on the resulting protein.** - **The existence of multiple repair systems highlights the importance of maintaining DNA integrity.** **Molecular Genetics((Ribonucleic Acid))** RNA RNA is a single stranded structure. Uracil substitute thymine. RNA sugar is ribose hydroxyl group of the 2' carbon position. RNA is used to transmit genetic information from the nucleus to cytoplasm where RNA is translated into peptides and proteins in ribosome. Transcription is process to copy DAN to RNA. - **Type of RNA:-** 1. Ribosomal RNA (rRNA) -- which makes up a large part of ribosomal structure on the endoplasmic reticulum in the cytoplasm. RNA polymerase I transcribes rRNA -- is found within the ribosome where protein synthesis occurs. 2. RNA (mRNA) -- A linear sequence of nucleotide which is transcribed from and complementary to a single strand of DNA and carries the information to ribosome to synthesis of protein. RNA polymerase II transcribed mRNA. 3. Transfer RNA (tRNA) transfers amino acids to the ribosome, where they are joined. - **Transcription** is the cellular process by which DNA is copied to RNA, where RNA is synthesized in a 5' to 3' direction. mRNA protected from denature by 7-methyl guanosyl cap added at 5' and adenines at 3'. RNA splicing is process to remove introns and remain exons on mRNA "mature" which code for peptides and proteins are joined together by process called ligation. mRNA mature transport out of nucleus. - **Translation** is the process by which RNA transcripts are turned into proteins and peptides. Translation is a complication process and involve three major steps:- 1. Initiation -- attachment of free Methionine to a transfer RNA molecule called (tRNA). 2. Elongation. 3. Termination, when ribosome move down the mRNA comes to one of the three stop codons UAA, UGA or UAG. The translation of that mRNA is finished and separate mRNA from ribosome. **Modern Genetics Techniques** **DNA Typing** Basic Techniques -- The steps are common to most techniques of molecular biology:- 1. Isolated material with minimum structure damage. 2. These must be away to visualize and locate the molecular species to be studied. 3. Method to separate out different species. 4. A method to quantify the isolated and studied species has to be used to get different exact as possible in the process studied. **DNA Isolation** Nucleic acid are isolated by alkaline denaturalization and precipitated with alcohol. Purified by use of specific columns that will bind nucleic acid based on change interaction. RNA requires greater care than DNA and must be used RNases. Complimentary DNA (cDNA) synthesis in RNA is converted into a DNA copy in vitro. Once DNA has been isolated, it can be stored in low salt buffers @-20°C on RNA @-80°C for many month or years. **Sequence Determination:** DNA analysis by:- 1. 2. - **Electrophoresis:** Comparison of DNA and RNA is often done by a process called gel electrophoresis. - **Cloning and PCR:** Cloning -- is away to make an exact copy of a desired DNA sequence and to obtain large amount of it in pure form. PCR -- is used to amplify isolated pieces of DNA or RNA. PCR use DNA polymerase and heating and cooling. **Important Points** - Genetics is defined as the study of inheritance or the transmission of characteristics from parents to off-spring. It is based on the biochemical structure of chromatin, which includes nucleic acids and the structural proteins that constitute the genetic material as well as various enzymes that assist in genetic processes such as replication. - All living organisms have specific numbers of chromosomes. Humans have 22 pairs of autosomes and one set of sex chromosomes, females (XX) and males (XY), giving a total of 46 chromosomes in diploid cells. - Mendel's law of independent assortment states that factors for different characteristics are inherited independent of each other if they reside on different chromosomes. - Human chromosomes are composed of the genetic material chromatin, a complex of the nucleic acid polymer DNA wrapped around highly basic proteins called histones. The helical structure of DNA allows a lot of information to be packaged in a very small amount of space. - Replication of DNA is semi conservative and is accomplished via the enzyme DNA polymerase, which produces a complementary duplicate strand of nucleic acid. Therefore, each strand of DNA can act as a template to be copied to make the opposite strand. DNA has a direction and is always read and written in the 5' (left) to 3' (right) direction. - Mutation refers to any structural alteration of DNA in an organism (mutant) that is caused by a physical or chemical agent (mutagen). Mutations can be beneficial or deleterious. Some mutations are lethal and therefore cannot be passed on to another generation. Some mutations are silent and have no consequence on the organism in which they occur and therefore have no selective pressure against them in the population. - Transcription is an enzymatic process whereby genetic information in a DNA strand is copied into an mRNA complementary strand. Eukaryotic mRNA is altered after it is made by various processing steps, such as the removal of introns and addition of a poly-A tail to the 3' end. These processing steps take place in the nucleus of the cell before the mRNA is exported to the cytoplasmic ribosomes for translation. - Translation is the complex process by which mRNA, which contains a mobile version of the DNA template encoding the genes for an organism, is turned into proteins, which are the functional units of an organism and the cells that it consists of. Translation occurs on the ribosomes, and additional steps may be necessary to get a specific protein into its final correct form, such as with the insulin molecule that requires disulfide linkages. Proteins are made of strings of amino acids and are always read in an amino terminal (left) to carboxyl terminal (right) direction. - Various methods have been developed to manipulate DNA; these make up the part of genetics known as genetic engineering. Southern blotting, sequencing, and cloning analyze DNA, whereas Northern blotting and RNase protection assays analyze RNA. Western blotting and immunoprecipitation as well as mass spectrometry and 2-D gel analysis are used to study proteins. Blotting and various biochemical assays are used to analyze carbohydrates and lipids. - Restriction endonucleases are enzymes found in various strains of bacteria and used in molecular techniques to cut DNA at specific sites. Restriction digests have a particular and unique pattern characteristic of the DNA that was used as a template. This is a very useful technique because mutations in DNA often destroy or create new restriction patterns. - A DNA probe is a piece of DNA having a sequence for the specific gene of study that can be labelled in various ways, such as with a radioactive substance or by fluorescent compounds, and visualized by autoradiography or a type of fluorescent imaging system. - The polymerase chain reaction (PCR) is an in-vitro method for enzymatic synthesis and amplification of specific DNA sequences using a pair of primers, usually short nucleotide sequences that hybridize to opposite DNA strands and flank the region of interest. Various modifications have made the PCR reaction more efficient and specific and allow more complex analysis. - A cloning vector is an extra-chromosomal genetic element that can carry a recombinant DNA molecule (bacteriophages, plasmids, or cosmids) into a host cell (bacteria, yeast, plant, or mammal). It can replicate on its own and may be very concentrated in a single cell. Most vectors contain markers for selection such as antibiotic resistance or fluorescent proteins. - Sources of DNA for analysis by molecular techniques may include any prokaryotic, nonnucleated cells, such as bacteria, or eukaryotic, nucleated cells, such as leukocytes, and may come from living or dead tissue, such as cell samples, cell cultures, or forensic material. Good sources of RNA include any active or dividing cell source, such as reticulocytes or fetal liver. Care must be taken to avoid RNA degradation by naturally occurring RNases. Nonactive cells often have low levels of mRNA, and greater care must be used to harvest sufficient good quality mRNA. Once the mRNA is isolated it can be transcribed into cDNA (complementary DNA) for further study by use of well-known reverse transcription techniques. - Many new techniques are now in use and are being developed for understanding the complete nature of the genetic material and how it functions in complex ways to control the nature of a living organism. This area of biology is growing very fast and will help to elucidate all the interactions, pathways, signals, and structures that make up the living cell and allow it to function in such a complex and sophisticated manner.