Different methods of gene isolation, Techniques of DNA sequencing,

Artificial DNA Synthesis

Mitesh Shrestha

Cloning strategies

DNA Sequencing

• Process of determining the precise order of nucleotides within a DNA molecule (As, Ts, Cs, and Gs).

• It includes any method or technology that is used to determine the order of the four bases—adenine, guanine, cytosine, and thymine—in a strand of DNA.

• The advent of rapid DNA sequencing methods has greatly accelerated biological and medical research and discovery.

Why sequence DNA?

• All genes available for an organism to use -- a

very important tool for biologists

• Not just sequence of genes, but also positioning

of genes and sequences of regulatory regions

• New recombinant DNA constructs must be

sequenced to verify construction or positions of

mutations

• Etc.

History of DNA sequencing

MC chapter 12

History of DNA sequencing

Technical Breakthrough For DNA Sequencing

In 1977, two separate methods for the

large-scale sequencing of DNA were

devised:

• Chemical cleavage method

by A. M. Maxam and W. Gilbert

• Enzymatic chain termination method

by F. Sanger et. al.

Of these two methods, Sanger method is more popular. Without changing the underlying concept of both methods, some improvements have been done over the years by applying different strategies, by developing various modifications and by automation. As a result, a very large scale sequencing has become feasible, e.g. E. coli, Saccharomyces cerevisiae, Human Genome Project etc.

Chemical Cleavage Method

• This method uses double-stranded DNA samples.

• Involves modification of the bases in DNA followed by chemical base-specific cleavage.

• Sequences DNA fragments containing upto ~500

nucleotides in length.

Stages: 1. The double-stranded fragment to be sequenced is isolated and radioactively labeled at the 5’-ends with 32P. 2. The fragment is then cut with restriction enzyme and thus the label is removed from one end. 3. The fragment of DNA with one end labeled is denatured. 4. Four identical samples of these end-labeled DNA restriction fragments are subjected to chemical cleavage at different chemical nucleotides. 5.There are four specific sets of chemical reactions that selectively cut the DNA backbone at G, A+G, C+T, or C residues.

– G only: dimethyl sulfate and piperidine – A+G : dimethyl sulfate and piperidine in

formic acid – C+T : Hydrazine, piperidine – C only : Hydrazine, NaCl, piperidine

Figure: Maxam-Gilbert method (continued) Lodish, H.;Berk, A. et. al. (4th ed); Mol. Cell Biol.; W. H. Freeman and Co. (2000) p: 233

6. For each labeled chain to be broken only once, the reactions are controlled.

7. The labeled subfragments created by the four reactions have

– the 32P label at one end and

– the chemical cleavage point at the other end.

8. The reaction products are separated by polyacrylamide gel electrophoresis which is based on size. Smallest fragment goes fastest. Figure: Apparatus for gel

electrophoresis

9. The labeled fragments

in the gel are visualized

by autoradiography.

10. The sequence is read

from bottom to top of

the gel.

Figure: Maxam-Gilbert method Lodish, H.;Berk, A. et. al. (4th ed); Mol. Cell Biol.; W. H. Freeman and Co. (2000) p: 233

Example of DNA Sequencing by Chemical Method

Mechanism of the chemical cleavage method

Voet, D.; Voet, J. Biochemistry; John Wiley and Sons, Inc (1990); p: 830

Continued

Voet, D.; Voet, J. Biochemistry; John Wiley and Sons, Inc (1990); p: 831

Advantages Disadvantages • No premature termination due

to DNA sequencing. So, no problem with polymerase to synthesize DNA.

• Stretches of DNA can be sequenced which can not be done with enzymatic method.

• Not widely used.

• Use of radioactivity and toxic chemicals.

Chain Termination method • This method uses single-stranded DNA.

• Also known as dideoxy sequencing method because it involves the use of analogue of normal nucleotide 2’,3’-dideoxynucleoside triphosphates (ddNTPs). These are chain terminating nucleotides lacking 3’-OH ends.

• This method is based upon the incorporation of ddNTPs into a growing DNA strand to stop chain elongation.

Figure: Structure of NTP, dNTP, and ddNTP

Lodish, H.;Berk, A. et. al. (4th ed); Mol. Cell Biol.; W. H. Freeman and Co. (2000), p: 233

Stages: 1.The DNA to be sequenced is called the template DNA. It is prepared as a single-stranded DNA

after being spliced into M13 vector DNA. Infected E. coli host cells release phage particles which contains single-stranded recombinant DNA that includes the sample DNA. This DNA sample is then extracted from phage for sequencing purpose.

2. A synthetic 5’-end-labeled oligodeoxynucleotide is used as the primer. 3. The template DNA is hybridized to the primer. 4. The primer elongation is performed in four separate polymerization reaction mixtures. Each

mixture contains - 4 normal deoxynucleotides (dNTPs) in higher concentration and - a low concentration of the each of the 4 ddNTPs. 5. There is initiation of DNA synthesis by adding enzyme DNA polymerase since the enzyme

cannot distinguish between the normal nucleotides and their analogues.

Figure: Action of DNA polymerase I

6. The strand synthesis

continues until a ddNTP

is added. The chain

elongation ceases on the

incorporation of a ddNTP

because it lacks a 3’-OH group

which prevents addition of the

next nucleotide.

7. There is a result of mixture of

terminated fragments, all of

different lengths.

8. Denature DNA fragments.

9. Each of the four mixtures are

run together on a

polyacrylamide gel for

electrphoresis.

Figure: Sanger method

Lodish, H.;Berk, A. et. al. (4th ed); Mol. Cell Biol.; W. H. Freeman and Co. (2000) p: 234

10. The separated

fragments are

then visualized

by autography.

11. From the position

of the bands of the resulting autoradiogram, the sequence of the original DNA template strand can be read directly.

Figure: Chain termination method Voet, D.; Voet, J. and Pratt, C. (upgrade ed) Fundamentals of Biochemistry; John Wiley and Sons, Inc (2002); p: 61

Advantages Disadvantages

• Most popular method.

• Simpler and quicker allowing large output. Within an hour the primer-annealing and sequencing reactions can be completed.

• Yielding of poor results owing to secondary structure in the DNA as sometimes DNA polymerases terminate chain elongation prematurely.

• The sequence is obtained not from the original DNA molecule but from an enzymatic copy. So, there is a chance of incorporation of wrong bases.

Example of DNA Sequencing in Sanger Method

http://users.wmin.ac.uk/~redwayk/lectures/sequence.htm

ddATP + ddA four dNTPs dAdGdCdTdGdCdCdCdG

ddCTP + dAdGddC four dNTPs dAdGdCdTdGddC

dAdGdCdTdGdCddC dAdGdCdTdGdCdCddC

ddGTP + dAddG four dNTPs dAdGdCdTddG

dAdGdCdTdGdCdCdCddG

ddTTP + dAdGdCddT four dNTPs dAdGdCdTdGdCdCdCdG

A

C G T

Chain Termination (Sanger) Sequencing

Cycle Sequencing

• Cycle sequencing is chain termination sequencing performed in a thermal cycler.

• Cycle sequencing requires a heat-stable DNA polymerase.

Fluorescent Dyes

• Fluorescent dyes are multicyclic molecules that absorb and emit fluorescent light at specific wavelengths.

• Examples are fluorescein and rhodamine derivatives.

• For sequencing applications, these molecules can be covalently attached to nucleotides.

Fluorescent Dyes

• In dye primer sequencing, the primer contains fluorescent dye–conjugated nucleotides, labeling the sequencing ladder at the 5′ ends of the chains.

• In dye terminator sequencing, the fluorescent dye molecules are covalently attached to the dideoxynucleotides, labeling the sequencing ladder at the 3′ ends of the chains.

ddA

ddA

AC GT

The fragments are distinguished by size and “color.”

Dye Terminator Sequencing

• A distinct dye or “color” is used for each of the four ddNTP.

• Since the terminating nucleotides can be distinguished by color, all four reactions can be performed in a single tube.

A

T

G

T

Capillary

G T C T G A

Slab gel

GA TC G A T C

Dye Terminator Sequencing

The DNA ladder is resolved in one gel lane or in a capillary.

• The DNA ladder is read on an electropherogram.

Capillary Slab gel

5′ AGTCTG

Electropherogram

Dye Terminator Sequencing

5′ AGTCTG 5′ AG(T/A)CTG 5′ AGACTG

T/T T/A A/A

Automated Sequencing

• Dye primer or dye terminator sequencing on capillary instruments.

• Sequence analysis software provides analyzed sequence in text and electropherogram form.

• Peak patterns reflect mutations or sequence changes.

Alternative Sequencing Methods: Pyrosequencing

• Pyrosequencing is based on the generation of light signal through release of pyrophosphate (PPi) on nucleotide addition.

– DNAn + dNTP DNAn+1 + PPI

• PPi is used to generate ATP from adenosine phosphosulfate (APS).

– APS + PPI ATP

• ATP and luciferase generate light by conversion of luciferin to oxyluciferin.

DNA sequence: A T C A GG CC T

Nucleotide added : A T C A G C T

Alternative Sequencing Methods: Pyrosequencing

• Each nucleotide is added in turn.

• Only one of four will generate a light signal.

• The remaining nucleotides are removed enzymatically.

• The light signal is recorded on a pyrogram.

Alternative Sequencing Methods: Bisulfite Sequencing

• Bisulfite sequencing is used to detect methylation in DNA.

• Bisulfite deaminates cytosine, making uracil.

• Methylated cytosine is not changed by bisulfite treatment.

• The bisulfite-treated template is then sequenced.

The sequence of treated and untreated templates is compared.

GTC

Methylated sequence: GTC Me

GGC Me

GATCTATC Me

GTGCA …

Treated sequence: Me

GGC Me

GATUTATC Me

GTGUA …

DNA Sequence:

(Untreated) reference: ...GTCGGCGATCTATCGTGCA…

Treated sequence: ...GTCGGCGATUTATCGTGUA…

This sequence indicates that these Cs are methylated.

Alternative Sequencing Methods: Bisulfite Sequencing

Next-generation sequencing (NGS)

• Also known as high-throughput sequencing, is

the catch-all term used to describe a number of

different modern sequencing technologies

including:

• Illumina (Solexa) sequencing

• Roche 454 sequencing

• Ion torrent: Proton / PGM sequencing

• SOLiD sequencing

Illumina (Solexa) sequencing

• In NGS, vast numbers of short reads are sequenced in a single stroke.

• To do this, firstly the input sample must be cleaved into short sections. The length of these sections will depend on the particular sequencing machinery used.

• In Illumina sequencing, 100-150bp reads are used. Somewhat longer fragments are ligated to generic adaptors and annealed to a slide using the adaptors. PCR is carried out to amplify each read, creating a spot with many copies of the same read. They are then separated into single strands to be sequenced.

• The slide is flooded with nucleotides and DNA polymerase. These nucleotides are fluorescently labelled, with the colour corresponding to the base. They also have a terminator, so that only one base is added at a time.

An image is taken of the slide. In each read location, there will be a fluorescent signal indicating the base that has been added.

• The slide is then prepared for the next cycle. The terminators are removed, allowing the next base to be added, and the fluorescent signal is removed, preventing the signal from contaminating the next image.

• The process is repeated, adding one nucleotide at a time and imaging in between.

454 sequencing

• Roche 454 sequencing can sequence much longer reads than Illumina. Like Illumina, it does this by sequencing multiple reads at once by reading optical signals as bases are added.

• As in Illumina, the DNA or RNA is fragmented into shorter reads, in this case up to 1kb. Generic adaptors are added to the ends and these are annealed to beads, one DNA fragment per bead. The fragments are then amplified by PCR using adaptor-specifc primers.

• Each bead is then placed in a single well of a slide. So each well will contain a single bead, covered in many PCR copies of a single sequence. The wells also contain DNA polymerase and sequencing buffers.

• The slide is flooded with one of the four NTP species. Where this nucleotide is next in the sequence, it is added to the sequence read. If that single base repeats, then more will be added. So if we flood with Guanine bases, and the next in a sequence is G, one G will be added, however if the next part of the sequence is GGGG, then four Gs will be added.

The addition of each nucleotide releases a light signal. These locations of signals are detected and used to determine which beads the nucleotides are added to.

This NTP mix is washed away. The next NTP mix is now added and the process

repeated, cycling through the four NTPs.

Ion Torrent: Proton / PGM sequencing

• Unlike Illumina and 454, Ion torrent and Ion proton sequencing do not make use of optical signals. Instead, they exploit the fact that addition of a dNTP to a DNA polymer releases an H+ ion.

• As in other kinds of NGS, the input DNA or RNA is fragmented, this time ~200bp. Adaptors are added and one molecule is placed onto a bead. The molecules are amplified on the bead by emulsion PCR. Each bead is placed into a single well of a slide.

• Like 454, the slide is flooded with a single species of dNTP, along with buffers and polymerase, one NTP at a time. The pH is detected is each of the wells, as each H+ ion released will decrease the pH. The changes in pH allow us to determine if that base, and how many thereof, was added to the sequence read.

The dNTPs are washed away, and the process is repeated cycling through the different dNTP species.

The pH change, if any, is used to determine how many bases (if any) were added with each cycle.

Improvements on the previous technology

• The four main advantages of NGS over classical Sanger sequencing are: – speed – cost – sample size – accuracy

• NGS is significantly cheaper, quicker, needs significantly less DNA and is

more accurate and reliable than Sanger sequencing. Let us look at this more closely. For Sanger sequencing, a large amount of template DNA is needed for each read. Several strands of template DNA are needed for each base being sequenced (i.e. for a 100bp sequence you'd need many hundreds of copies, for a 1000bp sequence you'd need many thousands of copies), as a strand that terminates on each base is needed to construct a full sequence. In NGS, a sequence can be obtained from a single strand. In both kinds of sequencing multiple staggered copies are taken for contig construction and sequence validation.

Improvements on the previous technology

• NGS is quicker than Sanger sequencing in two ways. Firstly, the chemical reaction may be combined with the signal detection in some versions of NGS, whereas in Sanger sequencing these are two separate processes. Secondly and more significantly, only one read (maximum ~1kb) can be taken at a time in Sanger sequencing, whereas NGS is massively parallel, allowing 300Gb of DNA to be read on a single run on a single chip.

• The reduced time, manpower and reagents in NGS mean that the costs are much lower.

Artificial DNA Synthesis

• Artificial gene synthesis, sometimes known as DNA printing is a method in synthetic biology that is used to create artificial genes in the laboratory.

• Currently based on solid-phase DNA synthesis, it differs from molecular cloning and polymerase chain reaction (PCR) in that the user does not have to begin with preexisting DNA sequences. Therefore, it is possible to make a completely synthetic double-stranded DNA molecule with no apparent limits on either nucleotide sequence or size.

• The method has been used to generate functional bacterial or yeast chromosomes containing approximately one million base pairs.

• Recent research also suggests the possibility of creating novel nucleobase pairs in addition to the two base pairs in nature, which could greatly expand the genetic code.