Chemical Synthesis, Sequencing and Amplification of Nucleic Acids

Question 1

What is chemically synthesized DNA/RNA?

Answer 1

Relatively short fragments of nucleic acids with defined chemical structure (sequence)

Question 2

In what direction does biosynthesis of DNA/RNA occur?

Answer 2

5’ to 3’ direction

Question 3

In what direction does the chemical synthesis of DNA/RNA of oligonucleotides occur?

Answer 3

The opposite direction - 3’ to 5’ direction.

Question 4

How is the chemical synthesis of oligonucleotides carried out?

Answer 4

Chemical synthesis is carried out using solid-phase synthesis using the phosphoramidite method

Question 5

What are phosphoramidites? And explain the phosphoramidite method.

Answer 5

• Phosphoramidites are formed when naturally occurring nucleotides (nucleoside-3’-or 5’-phosphates) are reacted with 3’-O-(N, N-diisopropyl-phosphoramidite) to form derivatives of nucleosides (nucleoside phosphoramidites).
• a phosphoramidite is a normal nucleotide but with protection groups, such as a trityl group, added to its reactive amine, hydroxyl and phosphate groups
• The phosphoramidite method uses building blocks derived from protected 2’-deoxynucleosides, ribonucleosides or chemically modified nucleosides
• This makes the nucleotides more reactive to expedite the synthetic preparation of oligonucleotides in high yields

Question 6

When the process of chemical synthesis is completed, what happens?

Answer 6

the product is released from the solid phase to a solution, de-protected, collected and purified using HLPC or PAGE; Dried

Question 7

To prevent undesirable side reactions in the chemical synthesis of DNA, what is done?

Answer 7

the functional groups present in nucleosides are made unreactive (protected) by attaching protecting groups

Question 8

What are the protecting groups used?

Answer 8

1) For the 5’ end - DMT
2) For the 3’ end -spacer linked to a controlled pore glass (CPG) bead

Question 9

How is oligonucleotide synthesis carried out?

Answer 9

by stepwise addition of nucleotide residues to the 5’-terminus of the growing chain until the desired sequence is assembled

Question 10

List the steps involved in oligonucleotide synthesis.

Answer 10

1. Deprotection
2. Coupling
3. Capping
4. Oxidation/Stabilization

Question 11

The column is washed each time with acetonitrile and purged with argon- why is this done?

Answer 11

to remove any unbound reagents and by products

Question 12

Explain the process of deprotection/ detritylation.

Answer 12

1. the trityl group (5’-DMT), which is attached to the 5’ carbon of the pentose sugar of the recipient nucleotide, is removed using 2% trichloroacetic acid (TCA) leaving a reactive hydroxyl group to which the next base is added
2. The DMT is washed away and the step results in the solid support-bound oligonucleotide precursor bearing a free 5’-terminal hydroxyl group

Question 13

Explain Coupling.

Answer 13

1. A 0.02 –0.2 M solution of nucleoside phosphoramidite solution and a catalyst tetrazole is added
2. The tetrazole activates the phosphoramidite so that the 3’-phosphite forms a covalent bond with the 5’-OH group. The mixing is usually very brief and occurs in fluid lines of the oligonucleotide synthesizer
3. Upon the completion of the coupling, any unbound reagents and by-products are removed by washing with acetonitrile and flushed with argon

Question 14

Describe Capping.

Answer 14

1. a small percentage of the solid support-bound 5’-OH groups (0.1 to 1%) remains unreacted and needs to be permanently blocked from further chain elongation to prevent the formation of oligonucleotides with an internal base deletion
2. The capping step is performed by treating the solid support-bound material with a mixture of acetic anhydride and dimethylaminopyrimidine as catalyst to acetylate the unreacted 5’-OH

Question 15

Stabilization/Oxidation

Answer 15

1. The newly formed internucleotide linkage is in the form of a phosphite triester linkage which is not natural and is not very stable under the conditions of oligonucleotide synthesis and is prone to breakage in the presence of acid or base
2. The treatment of the support-bound material with iodine and water in the presence of a weak base oxidizes the phosphite triester into a more stable pentavalent phosphate trimester. This is a protected precursor of the naturally occurring phosphate diester internucleosidic linkage
3. The column is then washed, dried and the cycle is repeated
4. When the final cycle is completed , the newly synthesized DNA strands are bound with CPG beads, each phosphate triestercontains a methyl group, every G, C and A carries an amino-protecting group and the 5’-terminus carries a DMT group

Question 16

Desalting is…

Answer 16

the process of removing contaminants in the oligonucleotide sequence.

Question 17

Why does desalting take place?

Answer 17

Because the protection groups that are removed from the oligonucleotide remain as organic salts.

Question 18

How is the process of desalting carried out?

Answer 18

• The 5’-terminus is phosphorylated with T4 polynucleotide kinase or chemically. Phosphorylation is done while the oligonucleotide is still bound to the support. All groups are then removed

Question 19

What are the applications of oligonucleotides in molecular biology and medicine?

Answer 19

1◦antisense oligonucleotides
2small interfering RNA
3◦primers for DNA sequencing
4◦primers for DNA amplification
5◦probes for detecting complementary DNA or RNA via molecular hybridization,
6◦targeted introduction of mutations and restriction sites,
7◦synthesis of artificial genes

Question 20

What is DNA Sequencing?

Answer 20

A technique that can determine the exact nucleotide sequence of a DNA molecule
lies at the core of genome analysis

Question 21

Complete the sentence: Knowledge of DNA sequences has become indispensable for -

Answer 21

basic biological research, and in numerous applied fields such as diagnostic, biotechnology, forensic biology, and biological systematics

Question 22

What does DNA replication in a cell involve?

Answer 22

1. DNA helicase
2. DNA polymerase
3. DNA template
4. Deoxynucleotides

Question 23

When does DNA replication start?

Answer 23

when DNA helicase unravels the double-helix structure to expose single-stranded DNA and form a replication fork

Question 24

Explain the process of DNA synthesis.

Answer 24

1. DNA helicase unravels the double helix structure to expose a single-stranded DNA and form a replication fork.
   2.A primer is introduced by RNA primase.
2. DNA polymerase then binds to the replication fork and starts DNA synthesis by sequentially adding nucleotides to the 3′-hydroxyl end of the RNA primer bound to the DNA template
3. The extension product grows in the 5′to 3′direction by forming a phosphodiester bridge between the 3´-hydroxyl group at the growing end of the primer and the 5´-phosphate group of the incoming deoxynucleotide

Question 25

What year was the Sanger dideoxy Sequencing/chain termination method introduced?

Answer 25

1974

Question 26

What does the Sanger process utilize?

Answer 26

it takes advantage of the ability of DNA polymerase to incorporate 2′, 3′-dideoxynucleotides- nucleotide base analogs that lack the 3′-hydroxyl group essential in phosphodiester bond formation

Question 27

What are the starting materials of the Sanger dideoxy sequence?

Answer 27

• DNA template
• sequencing primer
• DNA polymerase
• nucleotides (dNTPs)
• dideoxynucleotides (ddNTPs)
• reaction buffer

Question 28

How is the Sanger Sequencing Method carried out?

Answer 28

1. Four separate reactions are set up, each containing radioactively labeled nucleotides and either ddA, ddC, ddG, or ddT(32P)
2. The annealing, labeling, and termination steps are performed on separate heat blocks. DNA synthesis is performed at 37 °C, the temperature at which the T7 DNA polymerase used has the optimal enzyme activity
3. DNA polymerase adds either a deoxynucleotide or the corresponding 2′, 3′-dideoxynucleotide at each step of chain extension

Question 29

What determines whether a deoxynucleotide or a dideoxynucleotide is added to the chain in Sanger sequencing?

Answer 29

it depends on the relative concentration of both molecules.

Question 30

What happens when: a) a deoxynucleotide is added to the chain
b) a dideoxynucleotide added to the chain?

Answer 30

a) When a deoxynucleotide is added to the chain, chain extension can continue
b) chain extension terminates.

Question 31

In Sanger sequencing, how are products separated?

Answer 31

electrophoresis using polyacrylamide gels - products are separated by size (shorter strands move through the gel quicker).

Question 32

What are the starting materials for Cycle Sequencing?

Answer 32

• DNA template
• Sequencing primer
• thermal stable DNA polymerase
• deoxynucleotides (dNTPs)
• dideoxynucleotides (ddNTPs)
• reaction buffer.

Question 33

What is the key difference between the Sanger and Cycle Sequencing methods?

Answer 33

• Sanger uses radioactive material while cycle sequencing uses fluorescent dyes to label the extension products.
• components are combined in a reaction that is subjected to cycles of annealing, extension, and denaturation in a thermal cycler

Question 34

What are the advantages of performing cycle sequencing?

Answer 34

Protocols are robust, easy to perform, and effective for sequencing PCR products.

High temperatures reduce secondary structure, allowing for precise priming, template annealing, and thorough extension.

The same protocol can be used for double-and single-stranded DNA.

Difficult templates, such as bacterial artificial chromosomes (BACs), can be sequenced

Question 35

What is capillary electrophoresis?

Answer 35

Capillary electrophoresis is an analytical technique that separates ions based on their electrophoretic mobility with the use of an applied voltage

Question 36

What happens during capillary electrophoresis?

Answer 36

The extension products of the cycle sequencing reaction enter the capillary as a result of the electrokinetic injection.
A high voltage charge applied to the buffered sequencing reaction forces the negatively charged fragments into the capillaries. The extension products are separated by size based on their total charge

Question 37

The electrophoretic mobility of the sample can be affected by what?

Answer 37

1. Buffer type
2. Concentration
3. pH
4. The run temperature
5. Amount of voltage applied
6. Type of polymer used.

Question 38

How does detection occur in capillary electrophoresis?

Answer 38

• the fluorescently labelled DNA fragments, separated by size, move across the path of a laser beam.
• The laser beam causes the dyes on the fragments to fluoresce
• An optical detection device detects the fluorescence

Question 39

What are some applications of DNA sequencing?

Answer 39

De novo sequencing of genomes
Detection of variants (SNPs) and mutations
Biological identification (ancestry)
Confirmation of clone constructs
Detection of methylation events
Gene expression studies
Detection of copy number variation

Question 40

What are the types of DNA sequencing?

Answer 40

1. De novo sequencing
2. Shotgun sequencing
3. Next-generation sequencing
4. Whole genome sequencing

Question 41

Explain De novo sequencing.

Answer 41

The target DNA is fragmented and cloned into a viral or plasmid vector
Large-target DNA is cloned into Bacterial Artificial Chromosomes (BAC) with insert sizes ranging from 100 to 300kb.
The BACs are then subclonedinto smaller vectors that are more suitable for Sanger sequencing, with typical insert sizes of 1 to 10kb.
Bacterial clones are isolated and grown in media, the plasmid or phage DNA is extracted, and the purified DNA template is used for forward and reverse sequencing reactions.
Clones with small inserts can be completely sequenced using sequencing primers that hybridize to either end of the insert, then sequencing in the forward and reverse directions

Question 42

In De novo sequencing, why is cloning important?

Answer 42

Cloning is important because it provides amplification of the target DNA (by bacterial growth). It allows sequencing primers to bind to known sequences in the vector and extend the sequence into unknown target DNA.

Question 43

Explain shotgun sequencing.

Answer 43

Large target DNA is randomly cut into smaller pieces (0.5 to 1.5 kb) by enzymatic digestion
These shotgun fragments are subcloned into vectors, transformed into bacteria and isolated as colonies.
The colonies are inoculated into media and grown overnight.
The vector DNA is extracted and then sequenced from standard priming sites in the vector
- After sequencing individual fragments, the sequences can be reassembled on the basis of their overlapping regions

Question 44

What is Next Generation Sequencing (NGS)?

Answer 44

the catch-all term used to describe a number of different modern sequencing technologies

Question 45

What are the different types of NGS?

Answer 45

◦Illumina (Solexa) sequencing
◦Roche 454 sequencing
◦Ion torrent: Proton / PGM sequencing
◦SOLiD sequencing

Question 46

Advantages of Next Gen Sequencing are…

Answer 46

Rapidly sequence whole genomes
Zoom in to deeply sequence target regions
Utilize RNA sequencing to discovernovel RNA variants and splice sites, or precisely quantify mRNAs for gene expression analysis
Analyze genome-wide methylation or DNA-protein interactions
Study microbial diversity in humans or inthe environment