TUTORIAL: DNA BIOLOGY AND TECHNOLOGY 1. Describe the biochemical composition, structure and replication of DNA.

DNA is a polymer where the monomer units of DNA are nucleotides and the polymer is known as polynucleotide. DNA molecule consists of two strands of polynucleotide that wound together into a double helix. In the two polynucleotide strands is a phosphate group linked together by a phosphodiester bond between the phosphate group of one nucleotide and the hydroxyl group of carbon 3 of the next nucleotide that is 3’ terminal while at one end of each strand is a phosphate group linked to the carbon 5 of deoxyribose sugar. This is the 5’ terminal of each strand. The 3’ and 5’ terminals of the two strands are at opposite ends where the two strands is antiparallel. The two polynucleotide strands in the double helix DNA molecule are held together by hydrogen bonds between complementary purine-pyrimidine base pairs. There are four different types of nucleotides found in DNA, differing only in the nitrogenous which are adenine, guanine, cytosine and thymine. The sequence of steps in DNA replication is based on the semiconservative model that is takes place in the 5’-3’ direction using a DNA template strand. This process is catalyzed by DNA polymerase III. This process begin with the unwinding of the DNA double helix to form a replication fork. The unwinding is catalyzed by the enzyme, DNA helicase. The DNA helicase enzyme unwinds the double-stranded DNA to a replication fork ready for replication. The antiparallel nature of the strands, that is the 5’-3’ orientation of the top strand and the 3’-5’ orientation of the complementary bottom strand. Then, the DNA is already partially unwound to form a replication fork. On the bottom template strand, RNA primase attaches a short RNA primer in the 5’-3’ direction. After that, the RNA primase is removed, and free DNA nucleosides are added by DNA poltmerase III to the RM|NA primer in the 5’-3’ direction. This new strand is called the leading strand because it is made in the same direction as the movement of the replication fork. On the top template strand, RNA primase synthesizes a short RNA primer in the a short RNA primer in the 5’-3’ direction. RNA primase is removed, and DNA polymerase III adds nucleosides to the RNA primer one after another opposite to the replication fork

to form a short length DNA. This new short length is called the lagging strand because it is made in the direction opposite to the movement of the replication fork. The fragment produced is also called Okazaki fragment. The process repeat as the DNA continues to unwind. Then, the RNA primers are removed by a different type of enzyme called DNA polymerase I. The two Okazaki fragments are then sealed and joined up by the DNA ligase to produce a continuous chain called lagging strand antiparallel to the leading strand formed earlier. Two new antiparallel continuous strands which are one leading strand and one lagging strand, are formed. The new double-stranded daughter DNA molecules daughter DNA molecules then twist or wind to form double helix DNA molecules.

2.(a) Describe the steps in the synthesis of the polypeptide. There are two processes that undergoes by a cell in the synthesis of the polypeptide which are transcription and translation. Transcription is a process where the information in DNA is transcribed to a RNA molecule, called messenger RNA (mRNA). During transcription, one of the DNA strands acts as a template whereby a mRNA is transcribed complementary to the DNA template strand. A specific protein-coding gene consists of a promoter followed by the RNA-coding sequences for a protein and then a terminator. The promoter is a base-sequence that specifies where transcription is begins while the RNA-coding sequences is a base-pair sequence that includes coding information for the polypeptide chain specified by the gene. Besides, the terminator is a sequence that specifies the end of the mRNA transcript. Transcription is catalyzed by an enzyme called RNA polymerase when initiation of the transcription at the promoter site is begins. This involves specific recognition of promoter base sequence by RNA polymerase (in prokaryotes) or a complex of proteins (in eukaryotes). RNA synthesized is initiated with the addition of free RNA nucleotides in the 5’-3’ direction opposite with attachment at -OH end to the uncoiling direction of the DNA segment. The same rule of complementary base pairing are followed as in replication, except that uracil (U) replaces thymine (T) to pair with adenine (A), as RNA does not contain tnymine. After the RNA

synthesis is initiated, the RNA elongation will continue to the direction of 5’-3’ direction with the addition of more free nucleotides by RNA polymerase. Then, transcription will terminate when the terminator base sequence is recognized by RNA polymerase. In eukaryotes, introns and exons formed in the pre-mRNA during transcription are removed to produce mature mRNAs. While translation is a process where the genetic information transferred to a mRNA from the DNA is translated by ribosomes into amino acid sequence on polypeptides. This process requires the assistance of tRNA including the mRNA, rRNA and ribosome, enzymes and ATP which are initiated with the movement of mRNA from nucleus to ribosome. Ribosomes, the organelles on which the mRNA is translated, consist of two subunits, each of which contains rRNA an ribosomal protein. Ribosomes do not carry genetic information but facilitate the interaction of tRNA with mRNA during protein synthesis. The small subunit of a ribosome, binds to the 5’ end of an mRNA molecule. The small subunit slides along the mRNA until it reaches the start codon, which shows where translation should be initiated. The large subunit then binds to the small subunit to form a complete RNA-ribosome complex. The first tRNA occupies the P site and the A site is available for the next tRNA. At the end of the initiation stage, elongation begins when the vacant A site is bound by another charged tRNA with an anticodon complementary to the codon after the start codon on the mRNA. The ribosome moves three (a codon) nucleotides along the mRNA in a ‘5-3’ direction and detaches the peptide from the tRNA at the P site. The detached peptide is then attached by a peptide linkage to the single amino acid on the A site. In the process during which the ribosome slides across to the next codon of the mRNA, the tRNA at the P site is then displaced and detached from the ribosome. The P site is then adjacent tRNA carrying the polypeptide chain. The vacant A site will the be occupied by the next tRNA. The steps repeat in a cycle called ribosome until translation is terminated. When the ribosome reaches a stop codon, there is no tRNA that has a complementary anticodon. This signals the end of translation. The large subunit advances over the small subunit. The polypeptide is detached from the tRNA, and starts to fold up to form the final shape of the protein. The rRNA disintegrates to release the large subunit, small subunit, tRNA and mRNA.

(b) What would be the effect of a deletion or an addition in one of the DNA nucleotides Base deletion is the loss or removal of one or more nucleotides from a DNA nucleotide sequence while base addition is the insertion or addition of nucleotides into a DNA nucleotide sequence. When a deletion or an addition in one of the DNA nucleotides, it may alter a large portion of the resulting polypeptide. Base addition and deletion are also known as frameshift mutation. A frameshift mutation is a gene mutation that inserts or deletes a number of nucleotides (that are multiples of threes) from DNA nucleotide sequence. For example, the sequence given, TAC-GAA-CTT-CGG-TCC is inserted with a nucleotide base A and becomes TAA-CGA-ACT-TCG-GTC-C. This insertion or deletion can disrupt the reading, or the grouping of the codons where it can cause the codons to be read differently. Therefore, codons after the mutation will code for different amino acids. Furthermore, the stop codons (UAA, UGA, and UAG) will not be read, or a stop codon may be created at an earlier site. The protein created may be abnormally short, abnormally long and contain the wrong amino acids. It will be most likely not functional.

(c) What would be the effects of a substitution on one of the nucleotides? Base substitution ocuur when one nucleotide is exchanged with another nucleotide that has a different base. For example, TAC is exchanged with TAA. This mutation alters only one amino acid. This can lead for different DNA sequence, which will be transcribed into a different mRNA. Later, the altered mRNA will be translated into a polypeptide with a different amino acid sequence from the normal one. This mutation is also known as a missense mutation which mostly codes for a different amino acod. The most common example of a base substitution effect is sickle-cell anemia.

3. Explain how the lac operon was controlled in prokaryotes In eukaryotes, each gene is regulated separately. However, in prokaryotes such as bacteria, some genes are arranged in clusters and are regulated together. This clusters of gene are called operon, for example, the lactose operon found in E. coli. The lac operon is composed of three lac genes (lacA, lacY and lacZ) coding for different proteins, plus a promoter gene and an operator gene. A regulator gene nearby codes for a repressor protein that binds to the operator when lactose concentrations are low and effectively blocks RNA polymerase's access to the promoter. This can cause the transcription is blocked. When milk is consumed and lactose levels are high, the lactose binds to the repressor changing its shape and effectively removing its blockage of the promoter. Then, RNA polymerase can initiate transcription of the genes.

4. Describe the PCR process briefly. PCR or Polymerase Chain Reaction is a method to produce many copies of specific fragments of DNA in vitro. It resembles replication but is carried in a test tube. The tools required for PCR include a thermocycler, a test tube containing the DNA sample, the enzyme DNA polymerase, that is, Taq polymerase, primers and free DNA nucleotides. The PCR is done from the original small sample of DNA fragment (target DNA). Then, the DNA fragment is heated to break hydrogen bonds and separate the strands. Each fragment is then cooled and the primers is added, so that they will bind to the ends of the DNA. After that, the free DNA nucleotides are added nd the temperature is raised. When the replication is completed to give two new complementary DNA strands. Then, the processes are kept repeating to produce millions of copies of the target DNA.



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