Question

Name at least two DNA sequencing technologies and explain the principle.  

Explain why organisms undergoing rapid evolutionary change often contain relatively large numbers of mobile DNA elements, whereas once organisms settle into a stable evolutionary niche, most of these mobile elements are lost.

What are the dominating taxonomic groups of microorganisms during a year cycle of a lake? Why?

Answer 1

1 DNA sequencing is the process of determining the order of nucleotides adenine, thymine, cytosine and guanine along a DNA strand. There are different technogies for DNA sequencing. Two of them are being described below:

a.Chain termination methods- It is also known as Sanger sequencing. It is the process of selective incorporation of chain- terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication. This method begins with denaturation of the double stranded DNA. The single stranded DNA is then annealed to oligonucleotide primers and elongated using a mixture of deoxynucleotide triphosphates (dNTPs), which provide the needed arginine(A), cytosine(C), tryosine(T), and guanine(G) nucleotides to build the new double stranded structure. A small quantity of chain termianting dideoxynucleotides triphosphates for each nucleotide is included. The sequence will continue to extend with dNTPs until a ddNTP attaches. When a ddNTP is attached to the elongating sequence, the base will fluoresce based on the associated nucleotide. By convention, A is indicatd by green fluorescence, T by red, G by black and C by blue. A laser within the automated machine used to read the sequence detects a fluorescent intensity that is translated into a peak. When a heterozygous variant occurs within a sequence, loci will be captured by two fluorescent dyes of eual intensity. When a homozygous variant is present, the expected fluorescent colour is replaced completely by the new base pair's colour.

b. Shortgun sequencing- It is a technique for sequencing large DNA sequences such as entire genomes. It is a technique for determining the sequence of entire chromosomes and entire genomes based on producing random fragments of DNA that are then assembled by computers that order fragments by finding overlapping ends. Shortgun sequencin.g is when a genomic library is constructed by ligating random genomic DNA fragments into a vector, and then a randomly sequencing these clones. The sequence data are tehn assembled to contigs using computers that determine regions of overlap. Closing the gap between contigs can be done by screening for library clones that hybridize to probes from ends of previously identified contigs or by PCR amplifying with primers that anneal to the ends of two known contigs. The drawback of this approach is that a large number of reactions must be performed to find the correct pair of primers.