notesfromrgsbatch13.files.wordpress.com · 2013. 10. 30. · topics tested: polymerase chain...

Topics tested: Polymerase Chain Reactions & Restriction Enzymes

Mendelian Genetics DNA Transcription and translation

Gene Mutation

Biology PPA2 Notes

2 Biology PPA2 Notes

Compiled by Ragini Verma

polymerase chain reactions

Polymerase Chain Reactions are enzyme reactions to amplify a specific DNA segment whose two ends of the sequence are known. It allows one to obtain sufficient amounts of

the DNA region (more than a million copies) in order to work with it.

Step (1) Denaturation by Heat (30seconds) During this step, heat (over 90 degrees) separates the double stranded DNA into two

single strands.

Step (2) Annealing of Primers to target sequence (45seconds) Two oligonucleotide primers are used to “bracket” / mark out the ends of the target sequence (One primer complementary to each strand). Annealing takes place between

around 40 degrees and 65 degrees celcius

Step (3) Extension (45 seconds) Enzyme called Taq DNA Polymerase replicates the DNA strands. (72 degrees: the

enzyme is able to withstand the heat without denaturing) It begins the synthesis process at the regions marked by the primers. It synthesizes new double stranded DNA

molecules, both complementary to the original double stranded target DNA region, by facilitating the binding and joining of the complementary nucleotides that are free in

solution (dNTPs).

Extension always begins at the 3’ end of the primer making a double strand out of each of the two single strands. Taq DNA Polymerase synthesizes exclusively in the 5’ to 3’ direction. Therefore, free nucleotides in the solution are only in the 3’ end of the primers constructing the complementary strand of the targeted DNA sequence.

The above three steps make up 1 PCR cycle. The cycle repeats itself about 30 – 40 times, before the polymerase chain reaction is completed. By the end of the first PCR cycle, there are two new DNA strands identical to the original target. The whole PCR procedure takes around 0.5 to 3hours to complete. Needed: DNA template, 2 oligonucleotide primers, Taq polymerase, nucleotides, buffer containing Mg2+

Biology PPA2 Notes 3


Restriction enzymes Restriction Enzymes are a special class of naturally occurring proteins.

Restriction enzymes recognise very specific palindromic sequences of about 4 – 8 base pairs (e.g. TCGA or GAATTC)

Then, they break the phosphodiester bond on each of the DNA strands (or you can say cleave the DNA), within that specific sequence.

Note: The reaction is reversible through the action of DNA ligase Cohesive Ends and Blunt Ends The restriction enzyme cleavage may generate either cohesive ends or blunt ends Blunt Ends: Enzymes that cut at precisely opposite sites in the two strands of DNA generate blunt ends without overhangs

3’ Overhangs: The enzyme cuts asymmetrically within the recognition site such that a short single-‐stranded segment extends from the 3’ ends

5’ Overhangs: The enzyme cuts asymmetrically within the recognition site such that a short single-‐stranded segment extends from the 5’ ends

Restriction enzymes are useful in distinguishing gene alleles (like our PCR Taster experiment!)



Mendelian genetics Useful definitions (Mostly adapted from Kim Chia):

Alleles: alternative forms of a gene Heredity: the way genes transmit biochemical, physical, and behavioral traits from parents to

offspring Locus: the specific location of a gene or DNA sequence on a chromosome

Phenotype: observable characteristic Genotype: the actual pair of alleles present in an individual Gene: the heritable entity that determines a characteristic

Dominant: the allele that is expressed in the phenotype of the heterozygote Recessive: the allele that does not contribute to the phenotype of the heterozygote

Homozygote: individual with two identical alleles of a given gene Heterozygote: an individual with two different alleles of a gene

Heterozygous carrier: unaffected parents who bear a dominant normal allele that masks the effects of an abnormal recessive one. They carry the recessive allele, but do not express its

characteristics phenotypically. Selfing: the process during which 2 individuals from the same cohort (for instance, the F1

generation) cross among themselves Pure-‐breeding organisms: organisms that are homozygous for a particular trait, meaning that they have two identical alleles at a locus and thus will always pass that trait on to their offspring

when bred with other individuals that are also pure-‐breeding for that trait So! What is all this about? Basically, somatic (non-‐sex) cells have two sets of chromosomes-‐ one inherited from each parent of ours. So let’s say your dad has a red nose and your mum has a blue one, and you have got both alleles in you now! What colour will your nose be? One of the alleles could be dominant, and one could be recessive. Dominance is about the relationship between the alleles, in which one allele masks or totally suppresses the expression of the other allele. So if your dad’s red nose gene is recessive and your mum’s blue nose gene is dominant, you will get a blue nose! (Also, this means that you’re heterozygous, because you inherited two different alleles of the same gene) BUT-‐ what is happening if my nose is purple instead? This is known as incomplete dominance, which happens when both alleles of a gene pair are expressed in a heterozygote. No complete dominance or recessiveness is shown. Another example is when there’s a red flower + white flower giving you a pink flower! What if I get two observable characteristics? There’s also another headache called co-‐dominance, whereby both alleles of a gene pair are expressed in a heterozygote. Again, no complete dominance or recessiveness is shown. Time to look into blood groups! Example of co-‐dominance is the AB blood group.



A bit about FAMILY TREES (aka pedigree)! • Shows line of descent • Males represented by squares and females by circles • Trait under investigation is shaded, if expressed • Deduce gene is inherited

Multiple Alleles is when there are more than two alleles for one gene, in the whole population! E.g. there are so many blood group alleles for our blood group gene. Polygenic Inheritance is when two or more genes affect a single phenotype and have an additive effect. Eg. Skin colour, which is determined by three or more separately inherited genes. Genetic Variations Continuous Variation Discontinuous Variation No clear cut differences, differences intermediates between them

Has clear cut differences, with no intermediates between then

Produced by many genes (polygenes)/ many pairs of alleles

Produced by single pair of alleles

Affected by many environmental conditions Unaffected by environmental conditions E.g. Height, Skin colour E.g. Ability to roll tongue, blood group • Remember that the environment might also affect one’s phenotype (observable

characteristic) • For example, hydrangea flowers of the same genotype range from blue-‐violet to pink,

depending on soil acidity How are genetic variations formed?

Crossing over during synapsis in prophase I (shuffles the parts of each chromosome) Sexual reproduction: fusion of male and female gametes recombines the genetic material from each parent in new ways within the zygote

The Laws (VERY IMPORTANT!): Ø Law of Segregation: Each somatic cell of an individual carries two alleles at any

one locus. The alleles of a gene pair segregate (separate) from each other during anaphase I of meiosis, which occurs during the formation of gametes.

Ø Law of Independent Assortment: The separation of the alleles of one gene pair is independent of the separation of alleles of other gene pairs, resulting in the independent arrangement and separation of homologous chromosomes.

(It’d be useful to revise mitosis/meiosis at this point!) Monohybrid Crosses: A monohybrid cross is carried out between parents that differ in the alleles they possess for a particular gene (e.g. smooth seed crossed with wrinkled seed)

Ø The ‘parent’ seeds are referred to as ‘parental generation’ Ø The new baby seeds after them are referred to

as filial generation (F1 generation or offspring 1)

Ø The next generation is known as the resultant generation (F2 generation)



Dihybrid Crosses: Dihybrids are hetereozygotes that contain both alleles for each character (e.g. AaBb) When two dihybrids cross, it is known as a dihybrid cross (e.g. AaBb x AaBb) E.g of punnet square for dihybrid cross.

Taken from: http://www.youtube.com/watch?v=cvTt-‐azvHsA (helpful video!) Punnet Squares show you what chance the parents have of producing offspring with a certain characteristic.

RULES 1. Capital letter for dominant, lower case for recessive 2. Write the key (which letter is what) 3. If letters are specified in the question, follow that 4. Always put the capital letter in front of the small letter 5. Be tidy and make sure it’s easy to tell the difference between your capital and

small letters

If it’s a sex-‐linked question, you have to use X and Y. Just remember that XY is male and XX is female. So e.g. it can be like XRXr telling you that it’s a female tongue roller.



Dna Transcription and translation What is RNA?

• RNA is another nucleic acid that is chemically related (but not completely identical) to DNA

• RNA helps to express the information contained within the DNA mRNA (messenger RNA) carries the genetic code for proteins rRNA (ribosomal RNA) forms structural and functional components of ribosomes tRNA (transfer RNA) helps to incorporate amino acids into polypeptide chains This flow of genetic information is known as the Central Dogma DNA is in the nucleus of the cell and it cannot come out, therefore we need to make RNA, which can come out of the nucleus before it makes protein! TRANSCRIPTION:

1. Initiation: RNA Polymerase recognises and binds to the specific promoter sequence and initiates transcription

2. Elongation: Where RNA polymerase moves in a 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. Complimentary bases are assembled. (U instead of T)

3. Termination: Where RNA polymerase encounters a transcription terminator in the DNA (to signal the end of transcription), releases the complete mRNA, and dissociates from the DNA

Basically, if the DNA 3’ to 5’ is TACGGC, the mRNA sequence will be (in 5’ to 3’)

AUGCCG.

Useful video: http://www.youtube.com/watch?gl=SG&hl=en-‐GB&v=WsofH466lqk

GENETIC CODING: • Every group of three nucleotides (in sequence) is known as a CODON • Each codon specifies one amino acid (The genetic code is known as a

triplet) • The genetic code is unambiguous – Each codon specifies only a single

amino acid • The genetic code is degenerate – A given amino acid can be specified by

more than one codon, this is the case for 18 of the 20 standard amino acids

• The code contains start and stop codons that are necessary for initiating and terminating translation

• The code is commaless – Once translation of mRNA begins, the codons are read one after the other, with no breaks

DNA transcription RNA translation Protein



• The code is universal – It is used by almost all viruses, bacteria, archae and eukaryotes

TRANSLATION: Translation is the conversion of the mRNA codon sequences into an amino acid sequence (forming a polypeptide chain) using enzymes and ribosomes

1. Initiation: Where the ribosome binds to the mRNA 2. Elongation: Where amino acids (carried by tRNAs) are incorporated

into the polypeptide chain 3. Termination: Where the tRNA, polypeptide chain and ribosomes are

released from the mRNA Useful video: http://www.youtube.com/watch?v=5bLEDd-‐PSTQ



Genetic mutations

Mutation is the first step of evolution as it creates a new DNA sequence for a particular gene, creating a new allele. It is the inherent tendency of organisms to undergo change from one hereditary state to another.

Can occur when an allele of a gene changes, becoming another allele (Gene mutation)

Can occur when segments of chromosomes, whole chromosomes, or entire sets of chromosomes change (Chromosome aberrations)

Spontaneous mutations arise independently of any external stimulus, and may or may not confer a selective advantage to the mutant. However, background mutation rates are generally not high enough to bring about any major problems. The problematic ones are mutations caused by mutagens. Mutagens are agents that can damage the DNA of cells, leading to mutation rates higher than the natural rate. COMMON MUTAGENS Chemical

Chemicals that can alter the DNA of a cell Base analogs – chemicals that are similar to nucleotides and are mistakenly used in the formation of new but defective DNA

Intercalating agents – chemicals that interact and insert directly into the DNA of cells, leading to stretching of DNA and problems with DNA replication

Other chemicals may alter the structure and pairing of bases in a DNA strand

Radiation

Gamma radiation from radioactive substances (common radiation source!)

Gamma radiation is ionising, and produces free radicals in cells that damage DNA and chromosome structures

Ultraviolet light is also a form of radiation that is mutagenic. UV light is preferentially absorbed by DNA and can also lead to DNA damage

Biological

Many viruses contain DNA that insert themselves into chromosomes/ genomes of human cells, leading to mutations in the genes of the cell as the sequence of the DNA is altered with the insertion of foreign viral DNA

E.g. Human Papilloma Virus



So, er, how do mutations happen? Chemical/Biological/Radioactive mutagens that cause-‐ 1. Modifying of nucleotide bases

Ultraviolet light, nuclear radiation, and certain chemicals can damage DNA by altering nucleotide bases so that they look like other nucleotide bases

When the DNA strands are separated and copied, the altered base will pair with an incorrect base and cause a mutation

2. Breaking of phosphate backbone Environmental agents such as nuclear radiation can damage DNA by breaking the bonds between oxygen and phosphate groups



Mutations can also be caused by errors that occur when a cell copies its DNA in preparation for cell division. A nucleotide base may be inserted/substituted or deleted from the DNA sequence.

The simplest alteration is base substitution, in which one of the bases gets substituted for another. It’s known as point mutation. (The mutation is only at that ‘point’). This is USUALLY not too bad since only one base is affected, you see! Compared to the next one, which is really quite bad.

Ø Transition mutations: where a purine (A/G) substitutes for another purine, or a pyrimidine (C/T/U) for another pyrimidine

Ø Transversion mutations: where a pyrimidine substitutes for a purine or vice versa

Ø Missense mutations: where the new nucleotide alters the codon so as to produce an altered amino acid in the protein product, e.g. AGU (coding for serine) gets altered to AUU (coding for IIe)

Ø Nonsense mutations (um HAHAHAHA): where the new nucleotide changes a codon that specified an amino acid to one of the STOP codons (TAA/TAG/TGA), hence stopping the translation of the messenger mRNA transcribed from this mutant gene prematurely. The earlier in the gene that this occurs, the more truncated the protein product will be, and the more likely it is to be unable to function.

Ø Silent mutations: where the new nucleotide changes a codon, but the

amino acid still gets coded! (Why? Because most amino acids are encoded by several different codons!) E.g. if AGU gets changed to AGC, it’ll still code for serine J so no big issue here, yipeee! These mutations are called silent because there is no change to their product, so these mutations can’t be detected without sequencing the gene (or its mRNA)

The other two kinds of alteration are insertion and deletion, which happen when a base is inserted or lost from the DNA sequence. These are known as frameshift mutations. These are pretty bad because after such alterations, all the subsequent codons after the mutation site get altered as well! L



SICKLE CELL ANaEMIA (Example of mutation)

This is a genetic disease of red blood cell disorders Red blood cells are (normally) round (like the first picture) and they move through small blood tubes in the body to deliver oxygen

However, sickle red blood cells become hard, sticky and pointed. When these sickle red blood cells go through the small blood tube, they clog the flow and break apart, causing pain/damage and a low blood count, or anaemia.

!! WOWOWOWWOW WHAT EXACTLY HAPPENED HERE?! Sickle cell anaemia is actually a mutation that occurs in the gene that codes for one of the four polypeptides that form haemoglobin. A normal allele can mutate to form the recessive allele when one single nucleotide is substituted with another (ie. Adenine with Thymine). This results in a different amino acid being formed (ie. Val instead of Glu), and it abolishes the CTGAGG sequence recognised and cut by the restriction enzyme (Mstll). To diagnose sickle-‐cell anaemia, they test for the presence or absence of the cleavage site for Mstll. This disease then gets inherited as a Mendelian trait L but it’s recessive, remember!

notesfromrgsbatch13.files.wordpress.com · 2013. 10. 30. · topics tested: polymerase chain...

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