sequencing genes and genomes.ppt

Sequencing Genes and Genomes

By: Shimaa M. El-Gamal

The Methodology for DNA Sequencing

A- chain termination method:- (Sanger and colleagues in 1977)up to 96 sequences can be obtained simultaneously in a single run of a sequencing machine.

B- pyrosequencing :- invented in 1998, forms the basis to a massively parallel strategy that enables hundreds of thousands of short sequences to be generated at the same time.

Chain termination DNA sequencing

• single-stranded DNA molecules that differ in length by just a single nucleotide can be separated from one another by polyacrylamide gel electrophoresis, in a slab or capillary gel.

Gene Cloning and DNA Analysis (6th edition) 2010

Chain Termination sequencing outline


• a small amount of each of four fluorescent labeled dideoxynucleotides (ddNTPs—ddATP, ddCTP, ddGTP, and ddTTP) is added to the sequence reaction.

• several hundred nucleotides may be polymerized before a dideoxynucleotide is eventually incorporated

• The mixture is loaded into a well of a polyacrylamide slab gel, or into a tube of a capillary gel system

Reading Sequence results


Which DNA polymerase can be used in chain termination sequencing?

• It must have no exonuclease activity:-• The 5 →3 exonuclease activity changing the lengths of ′ ′

syntheized strands so that they no longer run through the polyacrylamide gel in the appropriate order.

• The 3 →5 exonuclease activity more importantly will ′ ′remove a dideoxynucleotide that has just been added at the 3 end, preventing chain termination.′


Taq polymerases used in sequencing

Klenow polymerase Sequenase

modified version of the DNA polymerase I enzyme from E. coli

modified version of the DNA polymerase encoded by bacteriophage T7

the 5 →3 ′ ′ exonuclease activity removed no exonuclease activity

low processivity (synthesize a relatively short DNA strand ~250bp)

high processivity (up to 750 bp)

Chain termination sequencing requires a single-stranded DNA template

1- M13 vector:- Cloned DNA fragments that are longer than about 3 kb are unstable in an M13 vector and can undergo deletions and rearrangements. This means that M13 cloning can only be used with short pieces of DNA.

2- Plasmid vectors:-- Double-stranded plasmid DNA, can be converted into single-stranded DNA by denaturation with alkali or by boiling, contaminated with small quantities of bacterial DNA and RNA, which can act as templates in the sequencing experiment.

- phagemid, a plasmid vector that contains an M13 origin of replication and which can therefore be obtained as both double- and single-stranded DNA versions (fragments up to 10 kb)

3- thermal cycle sequencing:-- similar to PCR, but just one primer is used and the reaction mixture includes the four dideoxynucleotides- the product accumulates in a linear fashion- used with DNA cloned in any type of vector


Types of primers used in sequencing• Primer roles:

A- DNA polymerase initiate synthesis from it.B- Determine the region of template will be sequenced

• The primer types:-1- universal primer, complementary

to the part of the vector DNA immediately adjacent to the point into which new DNA is ligated

2- Internal primer, a nonuniversal primer, designed to anneal at a position within the insert DNA


Pyrosequencing• does not require electrophoresis• more rapid than chain termination sequencing• Only able to generate up to 150 bp in a single

experiment• can be automated in a massively parallel

manner that enables hundreds of thousands of sequences to be obtained at once, perhaps as much as 1000 Mb in a single run.

• Much more quick than chain termination• the method of choice for genome projects.

Pyrosequencing detects the pulses of chemiluminescence

• ssDNA molecules as the starting material• No dideoxynucleotides added• based on the detection of released pyrophosphate (PPi)

during DNA synthesis, so Sequnce “read” as the reaction proceeds, as the addition of a deoxynucleotide to the end of the growing strand is accompanied by release of a molecule of pyrophosphate, which can be converted by the enzyme sulfurylase into a flash of chemiluminescence.

• easily automated.

• Each deoxynucleotide is added separately, one after the other, with a nucleotidase enzyme also present in the reaction mixture to rapidly degrade all incorporated deoxynucleotides before the next one is added.


Detection of chemiluminscence pulses

Massively parallel pyrosequencing

- Break DNA into fragments between 300 and 500 bp- each fragment is ligated to a pair of adaptorsAdaptor roles:1- enable the DNA fragments to be attached to small metallic beads. As one of the adaptors has a biotin label attached to its 5 end′ , and the beads are coated with streptavidin, to which biotin binds with great affinity, just one fragment become attached to each bead 2-provide the annealing sites for the primers for PCR

- Each DNA fragment amplified by PCR to obtain enough copies for sequencing, so the same pair of primers can be used for all the fragments.- PCR is carried out in an oil emulsion, each bead residing in its own aqueous droplet within the emulsion

- After PCR, the aqueous droplets are transferred into wells on a plastic strip so there is one droplet and hence once PCR product per well, and the pyrosequencing reactions are carried out in each well.


How to sequence a genome???

How to assemble the thousandsor perhaps millions of individual sequences

into a contiguous genome sequence?• shotgun approach

the genome randomly broken into shortfragments. The sequences are examined for overlaps and to build up the contiguous genome sequence.

• Clone contig approachinvolves a pre-sequencing phase, a series of overlapping clones is identified.


The shotgun approach to genome sequencing

• must identify overlaps between all the individual sequences.

• An error in identifying a pair of overlapping sequences could lead to missing out parts of the genome.

• mainly used with the smaller bacterial genomes, ex. Haemophilus influenzae

Haemophilus influenzae genome project, 1995

• its genome is 1830 kb• 28,643 chain termination

sequencing experiments were carried out to 19,687 clones

• 4339 were less than 400 bp, so they rejected.

• 24,304 sequences entered into a computer, which spent 30 hours analyzing the data. The result was 140 contiguous sequences, each a different segment of the H. influenzae genome.


How to close the gaps between the individual segments??

1- sequencing more of the sonicated fragments

- But six times the length of the genome had already been sequenced

2- Directly close the gap by hybridization analysis of a clone library prepared in a λ vector.

- The library probed with a series of oligonucleotides whose sequences corresponded with the ends of each of the 140 segments


Problems with shotgun sequencingThe major problem when applying shotgun sequencing to a large

genome is the presence of repeated sequences and the possibility that the assembled sequence “jumps” between two repeats, so part of the genome is misplaced or left out.

Using genetic or physical map to direct the assembly of the shotgun sequencing results (called directed shotgun approach) can solve this problem is case of large genomes.


The clone contig approach

• can provide an accurate sequence of a large genome that contains repetitive DNA.

• More work, longer time, and more money.• construct the overlapping series of cloned

DNA fragments, each cloned fragment is sequenced and the genome sequence built up step by step.

- Clone contig method requires 1- The cloned fragments should be as long as possible to

minimize the total number needed to cover the entire genome

2- A high capacity vector• The first sequenced chromosome was chromosome III

of Saccharomyces Cerevisiae, cloned in a cosmid vector , 29 fragments cloned with average size 10.8 kb.

• Sequencing of the much longer human genome required 300,000 bacterial artificial chromosome (BAC) clones.

How they can Assemble all of these into chromosome-specific contigs????

Clone Contig assembly can made by:

1- Chromosome walking

2- Rapid methods such as:a- clone fingerprintingb- repetitive DNA PCR or interspersedrepeat element PCR (IRE–PCR)c- sequence tagged site (STS) analysis

Chromosome Walking

A clone is selected at random from the library, labeled, and used as a hybridization probe against all the other clones in the library to identify the overlapping clones, then one of the overlapping clones labeled to used as a hybridization probe


Chromosome Walking

• it begins at a fixed starting point and builds up the clone contig step by step, and hence slowly, from that fixed point.

• only attempted when the contig is for a short chromosome and so involves relatively few clones, or when the aim is to close one or more small gaps between contigs that have been built up by more rapid methods.

2- Rapid methods for clone contig assembly

clone contig assembly do not use a fixed starting point and instead aim to identify pairs of overlapping clones

Clone Fingerprinting• based on the identification of sequence features that are shared

by a pair of clones. The simplest approach is to digest each clone with one or more restriction endonucleases and to look for pairs of clones that share restriction fragments of the same size, excluding those fragments that derived from the cloning vector.

• high possibility that two clones that do not overlap will share restriction fragments whose sizes are indistinguishable by agarose gel electrophoresis.


Repetitive DNA PCR or Interspersed

Repeat Element PCR (IRE–PCR)

• primers anneal within repetitive DNA sequences and direct amplification of the DNA between adjacent repeats


Sequence Tagged Site (STS)

• By search for pairs of clones that contain a specific DNA sequence that occurs at just one position in the genome under study, If two clones contain this feature, then clearly they must overlap

• STS is any short piece of DNA that occurs just once in the genome, and has been sequenced in an earlier project, so its easy to have specific primers for it


Using a map to aid sequence assembly

- The positions of STSs within the genome will have been determined by genetic mapping or physical mapping

- The shotgun assembly will be more accurate with the aid of genetic mapping or physical mapping

Genetic Maps

• obtained by genetic studies using Mendelian principles and involving directed breeding programmes for experimental organisms or pedigreeanalysis for humans

• genetic mapping of DNA sequences which display variability in the human population. Ex. DNA markers

DNA Markers• Single nucleotide

polymorphisms (SNPs), typed with short oligonucleotide probes

• Restriction fragment length polymorphisms (RFLPs), typed by Southern hybridization of restricted genomic DNA, or by PCR


• Short tandem repeats (STRs), also called microsatellites, short repetitive sequences of 1–13 nucleotides in length, linked head to tail. These sequences repeats between 5 to 20 times in the STR.

• Typed by carrying out a PCR using primers anneal either side of the STR, and then examining the size of the resulting product by agarose or polyacrylamide gel electrophoresis


Physical Maps

• locate the positions of specific sequences on a chromosomal DNA molecule.

• The loci that are studied can be genes or DNA markers, ex. expressed sequence tags (ESTs),which are short sequences obtained from the ends of (cDNAs)

Technique used in Physical Mapping

1- fluorescence in situ hybridization (FISH), a cloned DNA fragment is labeled with a fluorescent marker and then hybridized to a preparation of chromosomes immobilized on a glass slide.


2- mapping reagent, a collection of overlapping DNA fragments spanning the chromosome or genome that is being studied.


sequencing genes and genomes.ppt

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