BIOTECHNOLOGY - MICR 421

Chapter 5. Chemical Synthesis, Sequencing and Amplification of DNA

I. Chemical Synthesis of DNA
II. DNA Sequencing Techniques
III. Polymerase Chain Reaction

I.
Uses for Chemically Synthesized DNA
1. Labeled DNA probes to detect a target sequence
2. Primers needed for enzymatic biosynthesis of labeled probes and cDNA, DNA sequencing , and the polymerase chain reaction (PCR)
3. Mutagenesis. Altering a nucleotide sequence to introduce a mutation into a gene
4. Production of linkers and adapters for cloning
5. Synthesis of entire genes that cannot be cloned

Chemical Synthesis of DNA
(See Figs. 5.1 - 5.7)
Automated. Uses a DNA synthesizer
Phosphoramadites. Modified deoxyribonucleosides that are chemically coupled during synthesis
(See Fig. 5.3)
All steps are carried out while the DNA strand is bound to a column
(See FIg. 5.2)
Does not require a template strand of DNA, so you specify the sequence you need
Chemical synthesis proceeds in the 3' to 5' direction (remember that biosynthesis is in the 5' to 3' direction)
Not as efficient as DNA polymerase
The effiency of chemical coupling of phosporamadites affects the amount (yield) of the oligonucleotide that is produced.
See Table 5.1
Ex. Synthesis of a 20-mer with a coupling efficiency of 99%
0.9920 x 100 = 82% of product is full length of 20 bp
Long sequences are difficult to accurately synthesize

Synthesis of a labeled DNA Probe Using Based on the Amino Acid Sequence of the Gene Product
You know the protein you are interested in and want to use a DNA probe to screen a gene library to clone the gene. However, you have no clue as to what the sequence of the probe needs to be so that it will hybridize to the gene during library screening.
1.) Purify the gene product (protein)
2.) Determine sequence of 10 to 50 amino acids at the amino terminal end
Use an amino acid analyzer
Ex. Asn-Phe-Tyr-Ala-Trp-Lys-etc.
3.) Determine all possible coding sequences using the genetic code. There will be several possibilities because the code is degenerate (redundant).
4.) Chemically synthesize all possible combinations of oligonucleotides
(Fewer oliogos can be synthesized if the codon bias of the organism is known)
Use a DNA synthesizer and phosphoramadites
5.) Attach a detectable label
Ex. Use a kinase and AT32P to end label the 5' end of the oligos with radioactive phosphate
Compare this method for producing a labeled probe with the random primer method discussed in Chapter 4.

II. DNA Base Sequence Determination: Sanger Dideoxy Chain Termination Method
You have succeeded in cloning the target DNA, now you want to know its nucleotide (base) sequence.
Components of Sanger Sequencing Reactions (Fig. 5.17)
1. DNA to be sequenced (template)
2. Primer (How do you know what the sequence of the primer should be?)
3. dNTPs: one radiolabeled (Ex. dAT32P or 35S-dATP, for detection)
4. ddNTPs (Fig. 5.14)
5. DNA polymerase
 
Comparison of the chemical sturctures of:
Dideoxynucleoside triphosphate (ddNTP)
See Fig. 5.14 A.
Deoxynucleoside triphosphate (dNTP)
See Fig. 5.14 B
 
DNA biosynthesis is terminated after incorporation of a ddNTP. WHY?
See Figs. 5.15 and 5.16
 
Steps:
1.) Four reactions are run in separate tubes at the same time.
Each of the four tubes contains , dATP, dTTP, dCTP and dGTP, a radiolabeled dNTP such as dATP containing a 35S or 32P label, but only one ddNTP (a different one in each tube).
2.) Each reaction is analyzed by gel electrophoresis to separate the synthesized strands by their length.
An electric charge is generated across the sequencing gel. A cathode (negative electrode) is placed at the top of the gel and an anode (positive electrode) is placed at the bottom of the gel. Since DNA carries a net negative charge, the fragments will be repelled from the cathode and will be attracted to the anode.
Polyacrylamide (or agarose) is a polymer that forms a meshwork that causes larger fragments to move more slowly than the smaller fragments as they migrate from the cathode to the anode end of the gel.
3.) The position of the labeled DNA fragments in the gel is determined by autoradiography which produces a picture of the gel by exposing it to film.
See Fig. 5.18
The nucleotide sequence of the DNA is read from the bottom of the gel to the top.
The bottom of the gel corresponds to the 5' end of the DNA strand while the top of the gel corresponds to the 3' end.
A sequencing gel can be used to determine the sequence of a strand of DNA that is ~250-500 nucleotides long.
Most DNA to be sequenced is much longer, Ex. 1000 nucleotides for a typical E. coli gene.
Primer walking is used to determine the complete sequence.
(See Fig. 5.21)
 
Automated Sequencing With ddNTPs Containing Fluorescent Labels
· Each ddNTP is linked to different fluorescent dye
· The dyes of the separated fragments are excited with a laser beam causing them to emit light (fluoresce) which is detected
· Each dye fluoresces at a different wavelength
· Detection of the wavelength specific for dach dye identifies the ddNTP
· One sequencing reaction contains all ddNTPs in one tube and is analyzed in one gel lane by electrophoresis
· Cycle sequencing employs the polymerase chain reaction to produce the chain termination products
· Some instruments can analyze 96 samples per run
· Fluorescent primers rather than ddNTPs can also be used
o Must run 4 separate reactions as for Sanger sequencing
o The 4 reactions are combined and analyzed in 1 lane of an electrophoresis gel

III. The Polymerase Chain Reaction (PCR)
See Figs. 5.22 to 5.25
· Greatly amplifies the amount of target DNA
· Reaction mixture components
1. DNA containing the target
Templates for synthesis of complementary strands
May be genomic DNA or cloned DNA located within a vector
2. Taq polymerase
Thermostable DNA polymerase from Thermus aquaticus
Thermophilic bacterium that grow in hot springs at ~60-80 oC
3. Set of 2 primers that flank the region of DNA to be amplified
Needed to prime DNA synthesis by providing a 3'-hydroxyl group
Forward primer hybridizes to upstream region of one DNA strand
Reverse primer hybridizes to downstream region of the other strand
4. dNTPs
 
· Incubate at three different temperatures per cycle
~95oC to denature the target (make it single stranded)
~55oC to allow the primers to bind (hybridize, anneal) to the target
~75oC optimal temperature for polymerase activity of Taq
· Repeat ~30 X for ~1,000,000-fold increase over the amount you started with
· An instrument called a thermocycler is used to automate change of temperature
 
Some Uses for PCR
1. DNA fingerprinting for identification of individuals
Exs.
Forensic analysis of DNA from crime scenes
Determining paternity (relationship of offspring to mother or father)
 
2. Detection of low levels of DNA
Ex. Diagnosis of viral diseases such as AIDS and screening donated blood
 
3. Screening individuals for inherited mutations
Ex. Detection of genetic defects of a fetus while still in the womb
 
4. Taxonomy and study of microbial communities
Ex. Amplification of 16S rRNA (Prokaryotes) or 18S rRNA genes (Eukaryotes)
-Genus and species can be identified after determining the gene's sequence and comparing it to the sequence of known organisms
5. DNA sequencing (cycle sequencing, see above) and many any other uses

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Last updated: January 12, 2009 /jdh

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