Biology PPA2 (Term 3!)

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1 Biology PPA2 (Term 3!) -Compiled by Jing En- Polymerase chain reaction - In vitro* enzymatic reaction to amplify a specific DNA segment whose two ends of the sequences are known. - To obtain sufficient amounts of the DNA region to work with. *In vitro: experiments using components of an organism isolated from their usual biological surroundings. 2 synthetic oligonucleotides or primers are used to bracket the target region to be amplified. - One primer is complimentary to one DNA strand at the beginning; a second primer is complementary to the other DNA strand at the end of the target region. Typically cycles. 1. Denaturation of DNA template (increase in temp. to 94) 2. Annealing of primers (decrease in temp. to 64) 3. Extension of primers (increase in temp. to 72) Requirements: - DNA template - Primers: Short single stranded DNA fragments of nucleotides long designed based on the sequence flanking (beside) the region to be amplified. - Heat-stable DNA polymerase enzyme - Nucleotides Speed. Specificity (amplify specific fragment). Sensitivity (works when DNA quality is poor) Gel electrophoresis Undergo gel electrophoresis to separate the DNA fragments based on size. - If RE digested: 2 bands. If not digested: 1 band. - Heterozygous: 3 bands. Buffer solution: Provides ions to support conductivity, maintain the ph Runs from negative (black) to positive (red) because DNA is negatively charged. Loading dye: - To make sure that you do not over-run gel electrophoresis (track how fast DNA is migrating across agarose gel) - Contains glycerol to render DNA samples denser than the running buffer so the DNA samples will sink into the well. DNA is stained with ethidium bromide to be readily detected at high sensitivity, detected as visible fluorescence when gel is illuminated with UV light. Lower concentrations of agarose are used for separating larger DNA molecules, while higher concentrations are used for separating smaller DNA molecules. DNA ladder: It has known sizes of DNA fragments which are used to compare with unknown DNA to identify the size of the unknown. (act as a basis of comparison)

2 Primer Dimer It is a potential by-product arising from primer molecules hybridizing and attaching to each other because of strings of complementary bases. DNA polymerase amplifies the primer dimers and creates competition for PCR reagents, potentially inhibiting amplification of the DNA sequence targeted for PCR amplification. Restriction digestion/enzymes - Recognize specific sequences - Restriction enzyme cleavage occurs through the breaking of phosphodiester bond on each of the DNA strands Reversible through the ligation by DNA ligase. - Recognize and cleave DNA within particular palindromic sequences (4-8 nucleotides) - Results in STICKY ENDS (cohesive ends) or BLUNT ENDS. Mendelian genetics (alleles, sex linkage, dominance) - Unit factors in pairs - Dominance/Recessiveness - Random segregation: During formation of gamates, the paired unit factors separate randomly Law of Segregation (Monohybrid cross) The 2 alleles for each gene separate during gamete formation. (Segregation of alleles corresponds to the distribution of homologous chromosomes to different gametes in meiosis) Law of Independent Assortment (Dihybrid crossed unlinked) Alleles of genes on non-homologous chromosomes assort independently during gamete formation PLEIOTROPY: Several characteristics are affected by 1 gene - Results in the multiple symptoms of some diseases e.g. sickle-cell disease (sickle-cell anemia, sickle shape cell, clump together and clot blood vessels) POLYGENY: Several genes affect the expression of the same characteristic - Additive effect POLYGENIC INHERITANCE e.g. skin tone EPISTASIS: One gene affects the expression of another CONTINUOUS VARIATIONS No clear-cut differences with intermediates between Produced by many genes/pairs of alleles Affected by environmental conditions Height, skin colour DISCONTINUOUS VARIATIONS Clear-cut differences with no intermediates between them Controlled by single pair of alleles of gene Unaffected by environmental conditions Blood group, rolling of tongue NORM OF REACTION: Phenotypic range of a genotype influenced by the environment. Important for variation: May be able to evolve faster if conditions on earth change Sex-linked genes (genes carried by sex chromosomes) For a recessive sex-linked trait to be expressed:

3 - A female needs 2 copies of the allele whereas a male only needs 1 CARRIERS: capable of passing the recessive gene to their offsprings INCOMPLETE DOMINANCE: Both alleles of a gene pair are expressed in a heterozygote. The phenotype will be intermediate between the 2 characteristics. CODOMINANCE: Both alleles of a gene pair are expressed in a heterozygote. Both characteristics are expressed in the phenotype. MUTIPLE ALLELES: A single characteristic appear in different alleles, of which any 2 may occupy the same gene locus on the homologous chromosomes. Using test cross to determine unknown genotypes (cross with homozygous recessive) Transcription and translation Ribonucleic acid (RNA) serves as an intermediate role in the expression of information contained within the DNA. RNA Ribose has a hydroxyl group at the 2 position Pyrimidine Uracil (lacks methyl group at C5) DNA Lacks hydroxyl group at 2 position Pyrimidine Thymine mrna Carries the genetic codes for proteins trna helps incorporate amino acids into polypeptide chains rrna forms structural and functional components of ribosomes TRANSCRIPTION 1. INITIATION: RNA polymerase recognizes and binds to the specific promoter sequence and initiate transcription. 2. ELONGATION: RNA polymerase moves in the 3 to 5 direction along one strand of DNA (template strand), unwinding it as it goes to synthesize RNA in the 5 to 3 direction Complementary bases are assembled (U instead of T) 3. TERMINATION: RNA polymerase encounters a transcription terminator (signals the end) in the DNA, releases the completed mrna and dissociates from the DNA TRANSLATION The designation of different amino acids by mrna nucleotide sequence (CODON) is referred to as the genetic code.

4 - Unambiguous: Each codon only specifies one amino acid - Degenerate: A given amino acid can be specified by more than 1 codon - Contain start and stop codons - Commaless - Universal In eukaryotes, first amino acid inserted into polypeptide chains is methionine (Met) UAA, UAG, UGA are used as stop codons. 1. INITIATION: Ribosome binds to the mrna 2. ELONGATION: Amino acids (carried by trna) are incorporated into the polypeptide chain 3. TERMINATION: trna, polypeptide chain and ribosomes are released from the mrna Anti-codon Gene mutation Variations of gametes caused by: Meiosis - Crossing over during prophase I (shuffles the parts of each chromosome) - Independent assortment of chromosomes at metaphase/anaphase I (shuffle chromosomes) Fertilization: Random fusion of gametes from 2 individuals GENE MUTATION: when allele of a gene changes CHROMOSOME ABERRATIONS: when segments or whole chromosomes change Spontaneous mutations arise independently of any external stimulus. Background mutation rates are generally not high enough to bring about major problems. Mutagens are agents that can damage DNA of cells, leading to high mutation rates. CHEMICAL MUTAGENS Base analogs: Chemicals that are similar to nucleotides and are often mistakenly used in the formation of new but defective DNA. Intercalating agents: Interact and insert directly into the DNA, leading to stretching of DNA and problems in DNA replication. Radiation: Gamma radiation is ionizing and produces free radicals in cells which may damage DNA and chromosome structures. Other chemicals may alter the structure and pairing of bases in a DNA strand BIOLOGICAL MUTAGENS Many viruses contain DNA that insert into the chromosomes of human cells. This leads to mutations in the genes of the cell as the sequence of the DNA is altered with the insertion of the foreign viral DNA. E.g. Human Papilloma Virus Virus inserts its DNA into the apoptosis gene (which controls cell death) Mutation Cell death does not occur Cells continue to divide at alarming rates and cannot die CANCER!

5 ENVIRONMENTAL AGENTS UV light, nuclear radiation: (modifying nucleotide bases) Alter nucleotide bases to look like others. Altered base will pair with an incorrect base. Nuclear radiation: (breaking of phosphate backbone) Damage DNA by breaking bonds between oxygen and phosphate groups Errors that occur during DNA replication Base substitution (point mutation) Transition or transversion mutations Transitions: purine (A or G) substitutes for purine (A or G) or pyrimidine for a pyrimidine Transversion: pyrimidine (C or T) substitutes for a purine (A or G) or vice versa Missense Mutations New nucleotide alters the codon so as to produce an altered amino acid in the protein product. Nonsense Mutations New nucleotide changes a codon that specified an amino acid to one of the STOP codons Translation of the mrna transcribed from this mutant gene will stop prematurely. The earlier in the gene this occurs, the more truncated the protein product and the more likely it will be unable to function. Silent Mutations New nucleotide changes a codon but causes no change in their protein product Insertions and Deletions: Frameshift mutations which alter all subsequent codons downstream from mutation site Sickle Cell Anemia - Genetic disease of red blood cell disorders - Become hard and sticky, clog the flow and break apart. - Causes pain, damage and a low blood count, or anemia. A normal allele can mutate to form the recessive allele when one single nucleotide is substituted by another (A with T) in the middle position of codon 6 Converts GAG codon for Glu to a GTG codon for Val Hence abolishes the sequence (CTGAGG) recognized and cut by RE MstII.

6 Results in a different amino acid formed, changes sequence and hence characteristic of the polypeptide and protein. Molecular diagnosis of sickle-cell anemia involves testing for the presence or absence of a specific restriction enzyme (RE) cleavage site in DNA. Mutation that causes sickle-cell anemia removes a cleavage site for the RE MstII. Individuals with sickle-cell trait are immune to malaria parasite, Plasmodium falciparum, spread by mosquitoes. - Both are prevalent in West Africa, with heterozygous carrier individuals being less susceptible to malaria. - The sickle-cell allele has become prevalent in these populations due to selective pressure Mutations ANSWERING QUESTIONS 1. Name the mutation 2. Location - which nucleotide and codon 3. End result: Immediate (amino acid change) 4. Final result: Leads to the production of a non-functional protein. E.g. Point mutation/base substitution/transition mutation at 14 th nucleotide. Missence mutation alters codon from CGC to CAC. Hence altered amino acid from ARG to HIS in protein product, resulting in nonfunctional protein product. Primers: Provide the free 3 OH end for nucleotides to be added/elongated/to bracket the specific DNA region to be amplified. RE recognized specific restriction sites on a target DNA and breaks the phosphodiester bond. By using RE, you can distinguise the different alleles of depending on whether there is presence or absence of restriction sites. *ALWAYS BE SPECIFIC WHEN GIVEN CASE STUDIES. State allele e.g. Nn, state name of gene, state size e.g. 700kbp fragments Mendelian genetics Label if there are multiple punnett squares Always link back to the question! E.g.From the punnett square above, it shows that it is possible to produce a with heterozygous parents who are phenotypically identical. E.g. It is possible when both parents are heterozygous and are phenotypically identitical.