Name at least two DNA sequenci
Name at least two DNA sequencing technologies andexplain the principle.
Explain why organisms undergoing rapid evolutionarychange often contain relatively large numbers of mobile DNAelements, whereas once organisms settle into a stable evolutionaryniche, most of these mobile elements are lost.
What are the dominating taxonomic groups ofmicroorganisms during a year cycle of a lake? Why?
Answer:
1 DNA sequencing is the process of determining the order ofnucleotides adenine, thymine, cytosine and guanine along a DNAstrand. There are different technogies for DNA sequencing. Two ofthem are being described below:
a.Chain termination methods- It is also known as Sangersequencing. It is the process of selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitroDNA replication. This method begins with denaturation of the doublestranded DNA. The single stranded DNA is then annealed tooligonucleotide primers and elongated using a mixture ofdeoxynucleotide triphosphates (dNTPs), which provide the neededarginine(A), cytosine(C), tryosine(T), and guanine(G) nucleotidesto build the new double stranded structure. A small quantity ofchain termianting dideoxynucleotides triphosphates for eachnucleotide is included. The sequence will continue to extend withdNTPs until a ddNTP attaches. When a ddNTP is attached to theelongating sequence, the base will fluoresce based on theassociated nucleotide. By convention, A is indicatd by greenfluorescence, T by red, G by black and C by blue. A laser withinthe automated machine used to read the sequence detects afluorescent intensity that is translated into a peak. When aheterozygous variant occurs within a sequence, loci will becaptured by two fluorescent dyes of eual intensity. When ahomozygous variant is present, the expected fluorescent colour isreplaced completely by the new base pair’s colour.
b. Shortgun sequencing- It is a technique for sequencing largeDNA sequences such as entire genomes. It is a technique fordetermining the sequence of entire chromosomes and entire genomesbased on producing random fragments of DNA that are then assembledby computers that order fragments by finding overlapping ends.Shortgun sequencin.g is when a genomic library is constructed byligating random genomic DNA fragments into a vector, and then arandomly sequencing these clones. The sequence data are tehnassembled to contigs using computers that determine regions ofoverlap. Closing the gap between contigs can be done by screeningfor library clones that hybridize to probes from ends of previouslyidentified contigs or by PCR amplifying with primers that anneal tothe ends of two known contigs. The drawback of this approach isthat a large number of reactions must be performed to find thecorrect pair of primers.
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