DNA sequencing

sequencing

1. Microbial genetics
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DNA Sequencing

DNA sequencing, process by which the precise order of nucleotides in a piece of DNA
can be determined.

Methods of DNA sequencing
A. Radioactive methods
 The chemical degradation method (Maxam-Gilbert method), in which the
sequence of a double-stranded DNA molecule is determined by treatment
with chemicals that cut the molecule at specific nucleotide positions. Cleaves
DNA template at different nucleotide positions, G, A+G, T+C and C and labels
these cleaved fragments.Read A and T by interpreting double bands also
including G (with A) and C (with T) .Not used very much historically

 The chain termination method (Sanger dideoxy (enzymatic) sequencing)
in which the sequence of a single-stranded DNA molecule is determined by
enzymatic synthesis of complementary polynucleotide chains, these chains
terminating at specific nucleotide positions;
Amount of time and labor per sequence, cost of isotope, failed gels or failed
reactions, etc., result in hand sequencing being somewhat more expensive
to do than to do automated sequencing, and less reliable!

The term dideoxy comes from a special modified nucleotide, called a dideoxynucleotide
triphosphate (generically, a ddNTP). This modified nucleotide is key to the Sanger
technique because of its ability to block continued DNA synthesis. Because a
dideoxynucleotide lacks the 3’ hydroxyl group, this reaction cannot take place, and
therefore DNA synthesis is blocked at the point of addition. The logic of dideoxy
sequencing is straightforward.

Consider a segment of DNA that is about 1000 base pairs long that we wish to sequence.
(1) The two DNA strands are separated. Heating to 100˚C to melt the base pairing
hydrogen bonds that hold the strands together does this.
(2) A short oligonucleotide (ca. 18 bases) designed to be complimentary to the end of
one of the strands is allowed to anneal to the single stranded DNA.
(3) DNA polymerase is added along with the four nucleotide precursors (dATP,
dGTP, dCTP, and dTTP). The mixture is then divided into four separate reactions
and to each reaction a small quantity different dideoxy nucleotide precursor is
added. Dideoxy nucleotide precursors are abbreviated ddATP, ddGTP, ddCTP,
and ddTTP.
(4) The polymerase reactions are allowed to proceed and, using one of a variety of
methods, radiolabel is incorporated into the newly synthesized DNA.
(5) After the DNA polymerase reactions are complete, the samples are melted and
run on a gel system that allows DNA strands of different lengths to be resolved.
The DNA sequence can be read from the gel by noting the positions of the
radiolabeled fragments.
Obtaining the complete nucleotide sequence of a segment of DNA is often an important
part of understanding the organization of a gene and its regulation, its relation to other
genes, or the function of its encoded RNA or protein. Indeed, for the most part, translating
the nucleic acid sequence of a cDNA to discover the amino sequence of its encoded
polypeptide chain is simpler than directly sequencing the polypeptide itself.

2. Microbial genetics
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Thus dideoxynuclotides can be incorporated into DNA, but once a dideoxynuclotide has
been incorporated further elongation stops because the resulting DNA will no longer have
a free 3’ OH end. Each of the four reactions contains one of the dideoxynuclotides added
at about 1% the concentration of the normal nucleotide precursors. Thus, for example, in
the reaction with added ddATP about 1% of the elongated chains will terminate at the
position of each A in the sequence. Once all of the elongating chains have been
terminated there will be a population of labeled chains that have terminated at the
position of each A in the sequence.











B- Automated fluorescent sequencing
Cycle-sequencing in a thermal cycler, in one tube, with a commercially prepared mixture,
Still uses ddNTP technology of Sanger method, but with ddNTPs labeled with a different
colored fluorescent dye
ddATPs labeled with a green dye ,ddCTPs labeled with a blue dye
ddGTPs labeled with a black dye ,ddTTPs labeled with a red dye
Fragments electrophoresed on a polyacrylamide gel. As samples electrophorese across
a slit or window, a laser "reads" the fluorescent peaks representing different-sized
fragments Subsequent analysis of intensity peaks, and their colors, against a known
standard, gives the sequence.










Much less time and cost in labor, fewer failed reactions, fewer problem points for
"trouble-shooting" or optimization; cost of sequencing is largest expense, cost of cycle-
sequencing is next one, but tiny volumes used make this relatively cheap

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Suppose we have determined the nucleotide sequence of DNA fragment. How can we
tell whether it contains one or more genes? The nucleotide sequence is fed into a
computer, which then scans all six reading frames (three in each direction) in the search
for possible protein-coding regions that begin with an ATG initiation codon, end with a
stop codon, and are long enough that an uninterrupted sequence of its length is unlikely
to have arisen by chance. These stretches are called open reading frames (ORFs).
They represent sequences that are candidate genes.
Genome sequencing
A single chain termination sequencing experiment produces about 750 bp of
sequence, Clearly a large number of sequencing experiments must be carried out in
order to determine the sequence of an entire genome.
Two different strategies have been developed for sequence assembly (Figure ):
 The shotgun approach, in which the genome is randomly broken into short
fragments. The resulting sequences are examined for overlaps and these are
used to build up the contiguous genome sequence.
 The clone contig approach, which involves a pre-sequencing phase during which a
series of overlapping clones is identified. This contiguous series is called a contig.
Each piece of cloned DNA is then sequenced, and this sequence placed at its
appropriate position on the contig map in order to gradually build up the
overlapping genome sequence.

















Chromosomal Organization of Genes and Noncoding DNA
 In the genomes of prokaryotes and most lower eukaryotes, which contain few
nonfunctional sequences, coding regions are densely arrayed along the genomic
DNA.
 In contrast, vertebrate genomes contain many sequences that do not code for RNAs
or have any structural or regulatory function. Much of this nonfunctional DNA is
composed of repeated sequences. In humans, only about 1.5 percent of total DNA
(the exons) actually encodes proteins or functional RNAs.
 Eukaryotic genomic DNA consists of three major classes of sequences: genes
encoding proteins and functional RNAs, including gene families and tandemly
repeated genes; repetitious DNA; and spacer DNA

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• Next-generation” sequencing (NGS) is a high-throughput technology that
enables simultaneous sequencing of multiple DNA segments in a sample. This
analysis is accomplished by parallel sequencing and aligning these to a reference
sequence.
Different platforms use different sequencing approaches, NGS can be used to sequence
the entire complement of DNA in a sample (whole genome sequencing—WGS) or
specific segments can be isolated for sequencing. These can include all of the exons
(whole exome sequencing—WES), or specific regions of interest, creating “panels” of
genes to be sequenced.

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Metagenomics is the study of genomes from multiple organisms that inhabit a particular
environment. Metagenomics is an NGS applied to complex biological samples containing
several or many different organisms (mostly micro-organisms) as they are found in
environmental samples or stool probes.
In order to obtain the list of organisms present in the sample as well as their relative
abundance, either a genomic region of high variability flanked by well-conserved regions
is amplified (such as 16S ribosomal RNA fragments) or simply all DNA fragments present
in the samples are amplified. In the second case, alignment of the sequences with all
potential reference organisms is necessary.