Topic 5

Describe the structure of DNA and RNA

• DNA and RNA are formed from nucleotides that are linked together through a phosphodiester backbone in a linear direction

• Nucleotide: phosphate + sugar + base

• Nucleoside: sugar + base

What nitrogenous bases are purines?

• Adenine

• Guanine

What nitrogenous bases are pyrimidines?

• Cytosine

• Thymine

• Uracil

Describe the primary structure of DNA

• Found in all biological molecules

• Unique arrangement of deoxyribonucleotides or ribonucleotides arranged in a single chain

• Usually depicted as single letters in a row

Describe secondary structure of DNA

• Two complementary strands of DNA bind (anneal) together through complementary base pairing in an antiparallel fashion

• Resembles a double helix

A nucleotide structure consist of what?

• Nucleotide base (pyrimidine or purine)

• Ribose ring

• Phosphoryl group

What is the most abundant nucleotide?

ATP

ATP plays an important role in…

• Energy currency of metabolic pathways

• cAMP from ATP is one of the second messengers in cellular signal

Describe the primary level of DNA

Primary structure: single helix → nucleotides connected by phosphodiester bonds in a 5C → 3C direction

Describe the secondary level of DNA

• Secondary: double helix → both strands are antiparallel to each other

• 5’ → 3’

• Hydrophobic interactions, Van der Waals forces and hydrogen bonds

• Every turn in the helix have a 10.5 base pair

Describe the complementary base pairing

• Based on Chargaff’s rule

• States that A-T means A binds to T and G-C means that G binds to C

• These are called Watson-Crick base pairs

Why does supercoiling form?

Because of base stacking

Describe base stacking

• Bases are oriented so that hydrogen bonding with another base requires that they are arranged in a planar fashion, parallel to the adjacent bases on the same strand, and located in the interior of the helix

• The base pairs are stacked upon each other within van der Waals distance

• This provides stability to the molecule through the hydrophobic effect and van der Waals interactions

What are the forms of DNA?

• A-form

• B-form

• Z-form

Describe the A-form of DNA

• Short and wide

• Right-handed

• Dehydrated (cannot bind to water)

• Compacted form of DNA

• No major and minor grooves

Describe the B-form of DNA

• Most stable

• Right-handed

• Active DNA to make proteins

Describe the Z-form of DNA

• Most narrow

• Left-handed

• Compacted form of DNA

• It is elongated

• No major or minor grooves

Describe denaturation in DNA

• Also called “melting”

• Enzymes or chemicals

• Occurs under heating or addition of acid or base

• Separation into two individual strands

• Causes a “hyperchromic shift”

Describe renaturation

• Also called “annealing”

• Two strands reform a helix

• For PCR

• Requires lower temperature

Describe the melting temperature (Tm)

• It is where 50% is single strand and 50% is double strands

• Tm = ~80 to 85 Celcius

• The more double bonds are in a molecule, the higher Tm you need

Describe the hyperchromic shift

The difference in absorbance between double-stranded and single-stranded DNA after denaturation

Describe DNA supercoiling

• Structure where the majority of the DNA molecules inside a cell fold upon themselves

• The area where the double helix crosses itself

• Found in prokaryotes and eukaryotes

What are the types of supercoils?

• Positive supercoil

• Negative supercoil

Telomers have a high G-C content. What would be the optimal Tm of a telomere?

80 Celcius

Describe the nucleosome assembly

• Circular DNA is wrapped around histone proteins

• On turn must be removed in order for this wrapping to occur, which produces a negative supercoil, and which is balanced by adding a positive supercoil

Describe topoisomerases

• Enzymes that relive positive supercoils through cleavage and reannealing of DNA

• Type 1

• Type 2

Describe the type 1 topoisomerases

• Cleave one strand of DNA

• Reduce supercoiled region by one turn

Describe the type 2 topoisomerases

• Cleave two strands of DNA

• Reduce supercoiled region by two turns

Describe DNA

• Deoxyribose sugar

• Thymine base

Describe RNA

• Ribose sugar

• Uracil base

• Complex intrastrand structures

• Can form ribozymes (catalytic molecules)

Describe ribosomes

• RNA molecules with catalytic activity

• Ex: ribonuclease P (RNAse P)

• Cleaves nucleic acids

Basic function of tRNA

Help bring amino acids to the site of protein synthesis

Basic function of rRNA

Assembly of ribosomal subunit to make proteins

Basic function of mRNA

Help take genetic message from DNA to form proteins

Describe the unconventional base pairing in RNA and DNA

• Triplet interactions can occur between a single-stranded region of DNA, or RNA with an RNA, DNA, or RNA-DNA duplex

• This can result in a triple helix (triplex)

• Quadruplets can occur among guanine bases found in particular G-rich DNA sequences

• Intercalated motif (I-motif) structures consist of hydrogen bonds between hemiprotonated cytosine residues

Describe plasmids

• Self-replicating of circular DNA

• Can carry extra genetic information not contained in chromosomal DNA

• Are found in both prokaryotic and eukaryotic organism

• Contain an original of replication

• Can be cloned, conjugated, transformed, or transduced

What is the main DNA?

Chromosomal DNA

What is the acquired DNA?

Plasmid DNA

Describe conjugation

Two bacteria naturally come together and share DNA

Describe transformation

Environmental dependent

Describe transduction

Virus place bacteria into the bacteria

Describe endonucleases

• Enzymes that cleave

• Type 1

• Type 2

• Type 3

• Breaking DNA in the inside of the DNA double helix

• Specific hydrogen bonds between bases are broken

Describe type 1 and type 3 endonucleases

Require ATP

Describe type 2 endonucleases

• Cleave DNA at specific recognition sequences

• It is commonly used in labs

• Blunt and rough cutters

Describe blunt cutter type 2 endonucleases

• This will make a smooth, nice cut

• No overhang

Describe rough cutter type 2 endonucleases

• Will produce a 5’ overhang 

• Examples: EcoRI and Hpall

Describe EcoRI

• Recognize a specific base sequence

• 5’ GAATTC ‘3

• 3’ CTTAAG ‘5

Describe overhangs

• Also called “sticky ends”

• Easy to combine with ligases

Describe cDNA libraries

• Complementary DNA (cDNA)

• It is generated from mRNA by reverse transcriptase

• RNA fragments serve as primers

• DNA polymerases and DNA ligase aid in the generation of cDNA

• It is made without introns

Describe high-throughput DNA sequencing

• 1977 - Sanger sequencing developed

• Uses ddNTPs (dideoxynucleoside triphosphates)

• Generates chain termination DNA molecules

• Uses fluorescently labeled ddNTPs

• Followed by capillary gel electrophoresis

• 1980 - Nobel Prize awarded for DNA sequencing

Describe polymerase chain reaction (PCR)

• A method used to exponentially amplify a specific target DNA segment

• Number of DNA molecules increases by 2n in each cycle 

Why to use PCR?

• Amplifying DNA

• Make copies

• It can manipulate Tm

What are the three temperature phases of PCR?

• Phase 1 - DNA denaturation

• Phase 2 - Annealing

• Phase 3 - Primer extension and DNA synthesis

Describe phase 1 - DNA denaturation of PCR

• Break DNA/separate

• Tm is around ~95C

Describe phase 2 - annealing of PCR

• Annealing of primers

• Small sequences of DNA that act as a ladder sequence

• It has to match 1 strand on helix

• Cooling to around ~72C, so DNA primers can anneal to DNA template strand

Describe phase 3 - primer extension and DNA synthesis of PCR

Cooling to ~55C for DNA extension

PCR schematic (picture)

Polymerase chain reaction (picture)

• Step 1 is at 95C

• Step 2 is at 60C

Describe transcriptome analysis

• Gene expression microarrays

• Provide a readout of transcript abundance using a predetermined cDNA attached to a solid surface

• Next-generation sequencing using RNA sequencing

• Provides readout of all transcripts from same gene

• Permits identification of alternatively spliced RNA products

Describe CRISPR-Cas9

• An RNA-guided DNA targeting tool

• Discovered in the 1990s

• CRISPR = clustered regulatory interspersed short palindromic repeats

• A form of adaptive immunity based on specific recognition of bacteriophage DNA by complementary RNA that was transcribed

Based on the DNA produced in the gel, who was not at the crime scene?

The victim and suspect A

You perform a PCR experiment but add DNA polymerase instead of Taq polymerase. What will happen?

Replication will not occur