DNA SEQUENCING IN

PHYLOGENY

Bikash Kar NathMBM11005

M.Sc. 1st Semester. MBBT

Objectives of the Presentation

What is DNA sequencing?

What is Phylogeny?

DNA sequencing techniques.

Results of DNA sequencing.

Phylogenetic Interpretation of the results.

Construction of Phylogenetic Trees.

Applications.

Conclusion.

What is DNA sequencing?

DNA sequencing is a scientific approach involving various

biochemical, biophysical and computational techniques to

determine the order of the nucleotide bases- adenine, guanine,

thymine & cytosine in a molecule of DNA.

DNA sequencing techniques are key tools in many fields. A large

number of different sciences are receiving the benefits of these

techniques, ranging from archaeology, anthropology, genetics,

biotechnology, molecular biology, forensic sciences, among others.

What is Phylogeny?

According to modern evolutionary theory, all organisms on earth have descended from a common ancestor, which means that any set of species, extant or extinct, is related. This relationship is called phylogeny, and is represented by phylogenetic trees, which graphically represent the evolutionary history related to the species of interest.

Phylogenetic tree of various foxes

Common Ancestor

DNA Sequencing Techniques. Frederick Sanger’s Chain termination or Dideoxy-nucleotide Sequencing. Allan Maxam & Walter Gilbert’s Chemical Degradation Sequencing. Lynx Therapeutics' Massively Parallel Signature Sequencing. Polony sequencing. Pyrosequencing. Illumina sequencing. Solid sequencing. Ion semiconductor sequencing. DNA nanoball sequencing. Single Molecule SMRT sequencing &Single Molecule real time RNAP

sequencing. Nanopore DNA sequencing.

Sanger’s Dideoxy-nucleotide Sequencing.

ddGTPddATP ddCTP ddTTP

6. One type of ddNTP per reaction

7. 2. With region of known sequence

3’

8. ddNTP incorporation - stops chain synthesis

3’ 3’ 3’ 3’

3. Complementary primer

5’

1. Unknown fragment

5’

Reaction 1

5. Four separate reactions

3’ 3’ 3’

5’ 5’ 5’

5’ 5’ 5’

4. dNTP’s 35S labeled dATP or dCTP

DNA Polymerase

Reaction 4Reaction 3Reaction 2

(Read start be cloned for production of single-stranded DNA)

ddGTP ddATP ddTTP ddCTP

Sequence of unknown fragment

Shortest synthesized band = 5’ end of synthesized strand

Longest synthesized band = 3’ end of synthesized strand

3’

A

C

A

C

T

G

T

A

G

A

G

5’

Maxam & Gilbert’s Sequencing. The DNA to be sequenced must first be end labeled at one 5’ end generally by kinase

treatment with 32P ATP. Labeled DNA is first precipitated to remove any salts which might interfere in the cleavage

reactions. Chemical treatment generates breaks at a small proportion of one or two of the four

nucleotide bases in each of four reactions (G, A+G, C, C+T). The purines (A+G) are depurinated using formic acid, the guanines are methylated by DMS and the pyrimidines (C+T) are methylated using hydrazine. The addition of salt (sodium chloride) to the hydrazine reaction inhibits the methylation of thymine for the C-only reaction.

The modified DNAs are then cleaved by hot piperidine at the position of the modified base. The concentration of the modifying chemicals is controlled to introduce on average one modification per DNA molecule.

Thus a series of labeled fragments is generated, from the radiolabeled end to the first "cut" site in each molecule. The fragments in the four reactions are electrophoresed side by side in for size separation. To visualize the fragments, the gel is exposed to X-ray film for autoradiography, yielding a series of dark bands each corresponding to a radiolabeled DNA fragment, from which the sequence may be inferred.

Unknown DNA sequence

Next Generation DNA Sequencing Methods Massively Parallel Signature Sequencing :- MPSS was a bead-based method that used a complex

approach of adapter ligation followed by adapter decoding, reading the sequence in increments of four nucleotides. Subsequent development of less complex novel sequencing techniques made MPSS obsolete.

Polony sequencing :- It combined an in vitro paired-tag library with emulsion PCR, an automated microscope, and ligation-based sequencing chemistry to sequence an E. coli genome at an accuracy of > 99.9999% and a cost approximately 1/10 that of Sanger sequencing.

Pyrosequencing :-The method amplifies DNA inside water droplets in an oil solution (emulsion PCR), with each droplet containing a single DNA template attached to a single primer-coated bead that then forms a clonal colony. Pyrosequencing uses luciferase to generate light for detection of the individual nucleotides added to the nascent DNA, and the combined data are used to generate sequence read-outs.

Single Molecule real time RNAP sequencing:-This method is based on RNA polymerase (RNAP), which is attached to a polystyrene bead, with distal end of sequenced DNA is attached to another bead, with both beads being placed in optical traps. RNAP motion during transcription brings the beads in closer and their relative distance changes, which can then be recorded at a single nucleotide resolution. The sequence is deduced based on the four readouts with lowered concentrations of each of the four nucleotide types.

Results of DNA sequencing The sample sequence is

finally obtained as a graph with nucleotide peaks or as a false colour nucleotide base sequence.

Comparative analysis of DNA can be used as an important tool to analyze the phylogenetic relationship between organisms.

Electrophoregram

Multiple Sequence Alignment

Phylogenetic Interpretation of the results.

As obvious for a phylogenetic analysis DNA sequencing of more than a single species

and its subsequent sequence alignment is necessary.

Since the species supposedly share a common ancestry hence mismatches can be

interpreted as point mutations and gaps as indels (i.e. Insertion or deletion mutations)

introduced in one or both lineages in the time since they diverged from one another.

It is still a long and expensive process to sequence the entire DNA of an organism

(its genome) and this has been done for only a few species. However, it is quite feasible to

determine the sequence of a defined area of a particular chromosome. At any location

within such a sequence, the bases found in a given position may vary between organisms.

The particular sequence found in a given organism is referred to as its haplotype, and a

comparative analysis of these sequences can thus infer phylogenetic relationships.

Does a high degree of similarity mean that two DNA sequences have the same meaning or function?

“There are many scientists today who question the evolutionary paradigm and its atheistic philosophical implications”.

“There are not many scientists today who question the evolutionary paradigm and its atheistic philosophical implications”.

These sentences have 97% similarity and yet have almost opposite meanings!

Thus Homology among DNA is often incorrectly concluded on the basis of sequence similarity. The terms "percent homology" and "sequence similarity" are often used interchangeably. As with anatomical structures, similarity might occur because of convergent evolution (giving rise to homoplastic organs), similarly high sequence similarity may occur with shorter sequences, because of chance. Such sequences are similar but not homologous. Hence establishment of phylogenetic relationship takes into account conserved sequences occurring in all the sample species & its extent of conservation.

Construction of Phylogenetic Trees. To build phylogenetic trees, statistical methods are applied to determine the tree topology and calculate the

branch lengths that best describe the phylogenetic relationships of the aligned sequences in a dataset. Many

different methods for building trees exist and no single method performs well for all types of trees and

datasets.

Common approaches applied include the following:-

1. Distance-Matrix Methods:- They compute a matrix of pairwise “distances” between sequences that

approximate evolutionary distance.

2. Discrete data methods:- They examine each column of a multiple sequence alignment dataset separately

and search for the tree that best represents all this information. These methods produce a separate tree for each

column in the alignment, so it is possible to trace the evolution for specific elements within a given sequence,

such as catalytic sites or regulatory regions.

3. Maximum Likelihood:-The maximum likelihood method exhibits a probabilistic model of evolution for

estimating nucleotide substitution.

Thus a phylogenetic tree is

obtained highlighting the

relationship between various

species and the extent to

which they are related thereby

tracing their evolutionary

history.

Phylogenetic Tree of the Canid family

Applications

Tracing the evolution of Man.

Tracing the evolution of biologically vital proteins.

Tracing the evolution of infectious pathogens.

Increasing the efficacy & efficiency of drugs by sample testing on

phylogenetically related species.

DNA sequencing and its use in phylogeny are increasingly being used in

virology laboratories to study the transmission of viruses.

Conclusion

Establishment of phylogenetic relationship between various species

on the basis of DNA sequencing provides a detailed and reliable

approach to tracing evolutionary history of those species as well as

predicting their further evolutionary pattern. It is highly vital in

understanding spontaneously evolving viruses and infectious

pathogens & in an attempt to combat against their afflictions as well as

increasing the value of economically important organisms.

References A review of DNA sequencing techniques: Lilian T. C. Franc:a, Emanuel Carrilho and

Tarso B. L. Kist, Quarterly Reviews of Biophysics 35, 2 (2009), pp. 169–200. "

2009 Cambridge University Press.

Molecular Phylogenetics- An introduction to computational methods and tools for

analyzing evolutionary relationships: Karen Dowell

Information from various internet sources.

Thank You