Nucleic Acids - Los Angeles Mission College...• Nucleic acids are biopolymers made of nucleotides,...
Transcript of Nucleic Acids - Los Angeles Mission College...• Nucleic acids are biopolymers made of nucleotides,...
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Nucleic Acids
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• DNA and RNA are chemical carriers of a cell’s genetic information
• Coded in a cell’s DNA is the information that determines the nature of the cell, controls cell growth, division
• Nucleic acid derivatives are involved as phosphorylating agents in biochemical pathways
Nucleic Acids
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• Last, but not least of the 4 major classes of biomolecules to be introduced
• To introduce chemical details of DNA sequencing and synthesis
Why this Chapter?
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• Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), are the chemical carriers of genetic information
• Nucleic acids are biopolymers made of nucleotides, aldopentoses linked to a purine or pyrimidine and a phosphate
• RNA is derived from ribose • DNA is from 2-deoxyribose
– (the ' is used to refer to positions on the sugar portion of a nucleotide)
Nucleotides and Nucleic Acids
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• Adenine, guanine, cytosine and thymine are in DNA
• RNA contains uracil rather than thymine
Heterocycles in DNA and RNA
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• In DNA and RNA the heterocycle is bonded to C1 of the sugar and the phosphate is bonded to C5 (and connected to 3’ of the next unit)
Nucleotides
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Nucleotides (Continued)
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Nucleotides join together in DNA and RNA by as phosphate between the 5’-on one nucleotide and the 3 on another
One end of the nucleic acid polymer has a free hydroxyl at C3 (the 3 end), and the other end has a phosphate at C5 (the 5 end).
Nucleotides (Continued)
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• In 1953 Watson and Crick noted that DNA consists of two polynucleotide strands, running in opposite directions and coiled around each other in a double helix
• Strands are held together by hydrogen bonds between specific pairs of bases
• Adenine (A) and thymine (T) form strong hydrogen bonds to each other but not to C or G
• Guanine (G) and cytosine (C) form strong hydrogen bonds to each other but not to A or T
Base Pairing in DNA: The Watson–Crick Model
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• The G-C base pair involves three H-bonds
• The A-T base pair involves two H-bonds
Hydrogen Bonds in DNA
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• The strands of DNA are complementary because of H-bonding
• Whenever a G occurs in one strand, a C occurs opposite it in the other strand
• When an A occurs in one strand, a T occurs in the other
The Difference in the Strands
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• The strands of the DNA double helix create two continuous grooves (major and minor)
• The sugar–phosphate backbone runs along the outside of the helix, and the amine bases hydrogen bond to one another on the inside
• The major groove is slightly deeper than the minor groove, and both are lined by potential hydrogen bond donors and acceptors.
Grooves
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Nucleic Acids and Heredity
• Processes in the transfer of genetic information: • Replication: identical copies of DNA are made • Transcription: genetic messages are read and carried out of the
cell nucleus to the ribosomes, where protein synthesis occurs. • Translation: genetic messages are decoded to make proteins.
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• Begins with a partial unwinding of the double helix, exposing the recognition site on the bases
• When activated forms of the complementary nucleotides (A with T and G with C) associate, two new strands begin to grow
Replication of DNA
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• Addition takes place 5 3, catalyzed by DNA polymerase
• Each nucleotide is joined as a 5-nucleoside triphosphate that adds a nucleotide to the free 3-hydroxyl group of the growing chain
The Replication Process
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• RNA contains ribose rather than deoxyribose and uracil rather than thymine
• There are three major kinds of RNA - each of which serves a specific function
• They are much smaller molecules than DNA and are usually single-stranded
Transcription of DNA
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• Its sequence is copied from genetic DNA
• It travels to ribsosomes, small granular particles in the cytoplasm of a cell where protein synthesis takes place
Messenger RNA (mRNA)
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• Ribosomes are a complex of proteins and rRNA
• The synthesis of proteins from amino acids and ATP occurs in the ribosome
• The rRNA provides both structure and catalysis
Ribosomal RNA (rRNA)
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• Transports amino acids to the ribosomes where they are joined together to make proteins
• There is a specific tRNA for each amino acid
• Recognition of the tRNA at the anti-codon communicates which amino acid is attached
Transfer RNA (tRNA)
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• Several turns of the DNA double helix unwind, exposing the bases of the two strands
• Ribonucleotides line up in the proper order by hydrogen bonding to their complementary bases on DNA
• Bonds form in the 5 3 direction,
Transcription Process
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• Only one of the two DNA strands is transcribed into mRNA
• The strand that contains the gene is the coding or sense strand
• The strand that gets transcribed is the template or antisense strand
• The RNA molecule produced during transcription is a copy of the coding strand (with U in place of T)
Transcription of RNA from DNA
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• DNA contains promoter sites that are 10 to 35 base pairs upstream from the beginning of the coding region and signal the beginning of a gene
• There are other base sequences near the end of the gene that signal a stop
• Genes are not necessarily continuous, beginning gene in a section of DNA (an exon) and then resuming farther down the chain in another exon, with an intron between that is removed from the mRNA
Mechanism of Transcription
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• RNA directs biosynthesis of peptides and proteins which is catalyzed by mRNA in ribosomes, where mRNA acts as a template to pass on the genetic information transcribed from DNA
• The ribonucleotide sequence in mRNA forms a message that determines the order in which different amino acid residues are to be joined
• Codons are sequences of three ribonucleotides that specify a particular amino acid
• For example, UUC on mRNA is a codon that directs incorporation of phenylalanine into the growing protein
Translation of RNA: Protein Biosynthesis
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Codon Assignments of Base Triplets
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• There are 61 different tRNAs, one for each of the 61 codons that specifies an amino acid
• tRNA has 70-100 ribonucleotides and is bonded to a specific amino acid by an ester linkage through the 3 hydroxyl on ribose at the 3 end of the tRNA
• Each tRNA has a segment called an anticodon, a sequence of three ribonucleotides complementary to the codon sequence
The Parts of Transfer RNA
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The Structure of tRNA
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• As each codon on mRNA is read, tRNAs bring amino acids as esters for transfer to the growing peptide
• When synthesis of the proper protein is completed, a "stop" codon signals the end and the protein is released from the ribosome
Processing Aminoacyl tRNA
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• The order of the bases along DNA contains the genetic inheritance.
• Determination of the sequence is based on chemical reactions rather than physical analysis
• DNA is cleaved at specific sequences by restriction endonucleases
• For example, the restriction enzyme AluI cleaves between G and C in the four-base sequence AG-CT Note that the sequence is identical to that of its complement, (3)-TC-GA-(5)
• Other restriction enzymes produce other cuts permitting partially overlapping sequences of small pieces to be produced for analysis
DNA Sequencing
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• The Maxam–Gilbert method uses organic chemistry to cleave phosphate linkages with specificity for the adjoining heterocycle
• The Sanger dideoxy method uses enzymatic reactions
• The Sanger method is now widely used and automated, even in the sequencing of genomes
Analytical Methods
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• The fragment to be sequenced is combined with: A) A small piece of DNA (primer), having a sequence that is complementary to that
on the 3 end of the restriction fragment B) The four 2-deoxyribonucleoside triphosphates (dNTPs) • The solution also contains small amounts of the four 2,3-dideoxyribonucleoside
triphosphates (ddNTPs) • Each is modified with a different fluorescent dye molecule
The Sanger Dideoxy and Nucleotides
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• DNA synthesizers use a solid-phase method starting with an attached, protected nucleotide
• Subsequent protected nucleotides are added and coupled
• Attachment of a protected deoxynucleoside to a polymeric or silicate support as an ester of the 3 –OH group of the deoxynucleoside
• Step 1: The 5 –OH group on the sugar is protected as its p-dimethoxytrityl (DMT) ether
DNA Synthesis
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• Step 2: After the final nucleotide has been added, the protecting groups are removed and the synthetic DNA is cleaved from the solid support
• The bases are protected from reacting
DNA Synthesis: Protection
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• Step 2 (Continued): Removal of the DMT protecting group by treatment with a moderately weak acid
DNA Synthesis: DMT Removal
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• Step 3: The polymer-bound (protected) deoxynucleoside reacts with a protected deoxynucleoside containing a phosphoramidite group at its 3 position, catalyzed by tetrazole, a reactive heterocycle
DNA Synthesis: Coupling
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• Phosphite is oxidized to phosphate by I2 • The cycle is repeated until the sequence is complete
DNA Synthesis- Step 4: Oxidation and Cycling
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• All protecting groups are removed and the product is released from the support by treatment with aqueous NH3
DNA Synthesis- Step 5: Clean-up
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• Copies DNA molecules by unwinding the double helix and copying each strand using enzymes
• The new double helices are unwound and copied again
• The enzyme is selected to be fast, accurate and heat-stable (to survive the unwinding)
• Each cycle doubles the amount of material
• This is an exponential template-driven organic synthesis
The Polymerase Chain Reaction (PCR)
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• The subject DNA is heated (to separate strands) with
– Taq polymerase (enyzme) and Mg2+
– Deoxynucleotide triphosphates
– Two oligonucleotide primers, each complementary to the sequence at the end of one of the target DNA segments
PCR: Heating and Reaction
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• Temperature is reduced to 37 to 50°C, allowing the primers to form H-bonds to their complementary sequence at the end of each target strand
PCR: Taq Polymerase
• The temperature is then raised to 72°C, and Taq polymerase catalyzes the addition of further nucleotides to the two primed DNA strands
PCR: Annealing and Growing
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• Repeating the denature–anneal–synthesize cycle a second time yields four DNA copies, a third time yields eight copies, in an exponential series.
• PCR has been automated, and 30 or so cycles can be carried out in an hour
PCR: Growing More Chains