Bacterial genomes have between 1 and 6 million base pairs (Mb). Most plants and

Question

Bacterial genomes have between 1 and 6 million base pairs (Mb). Most plants and animals have about 100 Mb. Humans have approximately 2900 Mb. As a result, individual chromosomes may contain millions of base pairs. It is difficult to work with DNA sequences this large. As a result, the DNA is broken into smaller pieces (approximately 500 to 1000 bp each). These pieces are sequenced and then the sequenced pieces are examined and aligned based on overlapping sequence homology at their ends. By comparing the DNA sequences among organisms, scientists can determine: what parts of the genomes are most similar among organisms and therefore likely evolved earliest. what key changes exist in the genomes that may account for differences among related species, and what changes within species exist that may account for development of specific types of disease. The following activity has been designed to help you understand how genomes are sequenced and how the sequence information may be used. In 1980, Frederick Sanger was awarded the Nobel Prize for inventing the dideoxy method (or Sanger method) of DNA sequencing. A double-stranded DNA segment approximately 700 bp in length is heated (or treated chemically) to separate the two strands. The single-stranded DNA that results is placed into a test tube containing a 9-to-1 ratio of normal deoxynucleotides to dideoxynucleotides. A dideoxynucleotide has no -OH group at either the 2' or 3' carbon. As a whenever a dideoxynucleotide is added to the growing DNA strand, synthesis stops at that point. If the ratio of normal to dideoxynucleotides is high enough, where one will be included in the sequence is random.

Explanation / Answer

Polymerase Chain Reaction (PCR) generating thousands to millions copy of particular DNA sequence. In this method, we can add Deoxynucleotides, Taq DNA polymerase, Primers, milliQ water and your desired gene in a eppendorf tube. Four important reactions of PCR are: Initial denaturation, denaturation, annealing and final extension. You allow the replication to continue for the same length of time in each tube. After PCR reactions, the result will be analysed by agarose gel electrophoresis and you can dye the gel with ethidium bromide and observe the banding patterns on the gel. Further, the sequence of DNA will be identified through Sanger method. You can set up each of four test tubes as noted below

Tube Number

Deoxynucleotides

Dideoxynucleotides

1

dATP,dTTP,dGTP,dCTP

ddATP

2

dATP,dTTP,dGTP,dCTP

ddTTP

3

dATP,dTTP,dGTP,dCTP

ddGTP

4

dATP,dTTP,dGTP,dCTP

ddCTP

Tube Number

Deoxynucleotides

Dideoxynucleotides

1

dATP,dTTP,dGTP,dCTP

ddATP

2

dATP,dTTP,dGTP,dCTP

ddTTP

3

dATP,dTTP,dGTP,dCTP

ddGTP

4

dATP,dTTP,dGTP,dCTP

ddCTP

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