Ch. 21 Structure and Properties of DNA Flashcards
Primary Structure of DNA
- Left to Right: Phosphate, Sugar, Base
What are the differences between DNA and RNA?
- DNA
- Deoxyribose sugar (1 OH group)
- Double stranded
- Responsible for storing/transferring genetic info
- A-T, C-G
- RNA
- Ribose sugar (Has 2 OH groups)
- Single stranded
- Codes for amino acids; Acts as messenger between DNA & ribosomes to make proteins
- A-U, C-G
What’s the difference between a nucleoside and nucleotide?
- Nucleotide
- 5C deoxyribose sugar
- Phosphate group
- N-base
- Nucleoside
- No phosphate group (Just Base and sugar)
Which bases are pyrimidines? purines?
Pyrimidines: Thymine and Cytosine (& Uracil)
Purines: Adenosine & Guanine
What is the central dogma?
Genetic characteristics of organism encoded in DNA. These genes expressed by transcribing into mRNA, then translating amino acid sequences of the corresponding protein. Formed by Francis Crick in 1958, revised in 1970.
Replication->Transcription->Translation
Exceptions: no longer one gene, one protein hypothesis; aminoacyl tRNA synthetase: reads and makes amino acids
Explain the 1928 Griffith Experiment
Mice given 1) living virulent strain, 2) living avirulent, 3) heat-killed virulent, or 4) living avirulent+heat-killed virulent strain. Mouse 1 and 4 died. Developed transforming principle: avirulent able to “transform” into virulent. Discovered DNA, not protein, is genetic material.
Oswald Avery repeated principle in 1944.
Explain the Alfred Hershey and Martha Chase Experiment in 1952.
Experiment 1: Grew virus in radioactive sulfur. Sulfur labeled protein, not DNA because doesn’t contain sulfur. Labeled protein remains outside of cell, suggesting that protein NOT genetic material.
Experiment 2: Virus’ phosphorus (mostly found in DNA) labeled radioactive P32, so only DNA labeled. Labeled DNA enters cell to multiply. DNA IS genetic material.
What were James Watson and Francis Crick known for?
Discovery of double-helix structure in England 1953.
What was Rosalind Franklin known for?
Not being credited with the co-discovery of the double helical structure of DNA
Who discovered the double helical structure of DNA?
Watson, Crick, and Franklin
Describe the 4 main physical and chemical characteristics of DNA
- Double Stranded Helix
- Anti-parallel strands
- Complementary bases
- Hydrogen bonds between strands
How are sugars linked together?
Phosphodiester bonds that link 3’ carbon of sugar to the 5’ carbon of the next sugar
Adenine (Nucleobase)
Guanine Nucleobase
Cytosine Nucleobase
Thymine nucleoside
Uracil Nuclebase
What are the ribonucleosides and deoxyribonucleosides that correspond to the nucleobases:
A, G, C, T, U
Adenine: Adenosine; deoxyadenosine
Guanine: Guanosine; deoxyguanosine
Cytosine: Cytidine; deoxycytidine
Thymine: 5-Methyluridine; thymidine
Uracil: Uridine; deoxyuridine
How are Uracil and Thymine related?
Uracil undergoes methylation rxn via thymidine synthase (cofactor THF)
How many H bonds are between A-T?
2 H bonds
How many H bonds are between G-C?
3 H bonds (this makes it more stable than A-T)
What forms the backbone of DNA?
The phosphodiester bond; responsible for negative charge of DNA that stabilize DNA double helix by interacting with surrounding H2O
Which bases are the most frequently methylated?
Guanine and cytosine (about 70% GC base pairs methylated in mammals)
What is the Dam methylase?
Adenine contained in any GATC sequence
Dem methylase
Cytosine in sequence CCAGG
What does methylation do?
Inactivates DNA expression, protecting it from restriction endonucleases
Evolutionary advantage developed by bacteria. Needed to survive from bacteriophage, which normally destroys host DNA to replicate its own.
What is genomic imprinting?
different methylation pattern in maternal/paternal chromosomes at CpG nucleotides (promoter sequences: TATA, CG, CAAT)
Diseases associated: Prader-Willi/Angelmann’s Syndrome; Beckwith-Wiedmann Syndrome
What is an example of good methylation?
Women’s cells randomly pick one of their X chromosomes and shut it off (barr bodies). Both men and women only have one working X chromosome at a time. The woman can have either the maternal or paternal X turned off. This creates the mosaic female.
What is an example of bad methylation?
Fragile X syndrome. Fragile X Mental Retardation gene 1 (FMRI) is a regulatory site (CpG island). Normally not methylated so can use Fragile X Mental Retardation Protein when needed, but in Fragile X, it is. Cell unable to copy info from FMR1 gene, no mRNA, FMRP not made.
How can being a mosaic female help or hurt the Fragile X syndrome?
Being mosaic female can help if majority of X chromosome with methylated Fragile X gene is turned OFF. Severity mainly depends on how much good FMR1 gene is turned off.
What is the definition of a base analogue?
Can be substitded for normal nucleic acids
Acyclovir is a base analog of _____? Function?
Analog of guanine (guanosine without cyclic form)
Used for herpes virus infection
AZT is a base analog for ______? Function?
3’ deoxy 3’ azidothymidine analog of thymine
Used to treat Herpes simplex virus and HIV
6-Mercaptopurine is a base analog of
Analog of hypoxanthine (purine analog)
Used to treat acute leukemia
5 Fluorouracil is base analog of ___? Function?
Analog of thymine
Antimetabolite irreversible inhibitor of thymidylate synthetase (Used to inhibit DNA synthesis, esp in cancer)
2 amino purine is a base analog of? Function?
Analog of adenine
Can pair with T or C during DNA replication (mutagenic for bacteria, non/weakly mutagenic for eukaryotes)
5 bromouracil is a base analog of? Function? (NOT IN SLIDES)
Can pair with adenine or guanine (pyrimidine analog)
Used to treat neoplasms
Who were the two main people who published methods about DNA sequencing in 1977?
Chemical method: Maxam
Biochemical method: Sanger
Explain the Dideoxy Chain Termination DNA Sequencing Method
Always read from 5’-3’ (bottom to top)
What are the rules that explain complementary bases?
Chargaff’s rules #A=#T and #C=#G
The bases of DNA are hydro_____ while the phosphodiester bonds are hydro____.
base= hydrophobic, phosphodiester bond=hydrophilic
For the B form:
There are __ bp per turn
__ nm diam
__ nm gap between bases
__ nm makes a complete turn
- 5 bases per turn
- 0 diam
- 34 gap
- 4 nm makes complete turn
What is the purpose of the major groove?
Makes space for DNA binding proteins with bind via H bonds to bases
Which is the most common form of DNA helix?
B form
Describe the A form of DNA.
Similar to B. Right handed, but more compact and dehydrated. Bases tilted 20 degrees relative to helical axes. 0.23 nm between bases
11 bases per turn
Describe the Z form of DNA
LEFT handed, ZIG ZAG structure, forms under high salt concentration and presence of divalent cations; 0.38 nm between bases; not much difference between major and minor grooves
12 bases per turn
What are intercalating agents? Examples?
Hydrophobic heterocyclic ring Inserted between stacked base pairs
Distorts DNA helix, interferes with replication, transcription, and repair
Ex: Ethidium bromide (fluorescence), acridine orange (fluorescence), actinomycin D (interfere with replication/transciption for anti-cancer)
What is a plasmid DNA?
Circular DNA molecules (many found in bacteria), containing few-hundreds of genes
Many include antibiotic resistance genes
What are supercoils?
What does it mean to be negatively supercoiled?
Positively supercoiled?
Tertiary structures of DNA
Negative: Underwound (turn in the same direction)
Positive: Overwound (turn in opposite direction)
What factors contribute to denaturation of DNA?
pH <2.3 (protonation of bases, disrupts base pairing) or >11.5 (deprotonation of bases,, destroy H bond)
Temperature: causes DNA melting
Ionic strength: can disrupt the H bond (lower ionic strength= lower melting temp)
Lower GC content means less strong= lower melting temp
What is Tm defined as?
Melting temperature
Midpoint of absorbance increase (temp when 1/2 double stranded DNA converted to single stranded)
What is renaturation of DNA called?
Reannealing
What determines the length of time required for reannealing?
Depends on length and DNA sequence (longer and complex means takes longer to reanneal)
Slow compared to denaturation process
What is the renaturation of DNA used for?
- Determine frequency of certain sequences
- Locate specific base sequences
- Detect particular RNA species
Where are short, highly repetitive sequences found?
Telomeres (end) and centromeres (site of attachment to spindle fiber)
What is chromatin?
DNA (-) + histones (+)
What are histones?
Basic proteins containing + charged lysines and arginines
What are the 5 major classes of histones?
H1, H2A, H2B, H3, H4
What are nucleosomes?
Beadlike structures from resting nuclei
What are the steps of DNA packaging?
- Neg. charge DNA wraps around (+) charged histone protein
- Coil nucleosome beads into nucleoprotein fibers (chromatin fibers)
- Condensed into chromosomes
What is the difference between euchromatin and heterochromatin?
Euchromatin: less densely packaged (active genes expressed)
Heterochromatin: More tightly packed (decreased expression of genes)
What are the two major types of repetitive DNA sequences?
Tandem (repeated in one area of DNA) and Interspersed (repeated sequence scattered throughout genome)
What are the 3 classes of DNA sequences?
Highly repetitive (10-15% of mammalian DNA). Includes tandem repeats
Moderately repetitive (25-40% of mammalian DNA). Includes interspersed repeats.
Single copy (very low #): 50-60% mammalian DNA (i.e. coding regions)
What are the 3 subclasses of tandem repeats?
Satellites, minisatellites, microsatellites
What are the 2 subclasses of interspersed repetitive DNA sequences?
Short interspersed nuclear elements (SINE) - example Alu
Long interspersed nuclear elements (LINE)
What is Fragile X Syndrome?
How do we diagnose it?
Most common cause of inherited mental retardation. Seen more commonly in males.
fragile x gene (FMR1) contains tandemly repeated CGG sequence near 5’ end. Normally 6-50. Premutation=50-200. Full mutation >200 repeats.
At >230 CGG repeats, FMR1 gene found heavily CpG methylated, blocking transcription and silencing FMR1 (no FMRP made).
PCR and Southern blot
What are the techniques for recombining DNA?
Purification of Nucleic Acids
Enzymatic manipulation
DNA cloning
Analysis of DNA
Expression of Recombinant Protein
Site-directed mutagenesis
Purification of DNA: isolating total genomic DNA
- Isolation of total genomic DNA
- Lysis cell and nucleus with detergent (SDS) or lytic enzymes
- Degrade DNA bound protein with any nuclease with Proteinase K
- Extract DNA using column (or phenol/chloroform extraction method)
- DNA precipitation with ethanol
- Dry and redissolve in aqueous buffer
Enzymatic manipulation of DNA
- DNases/RNases break phosphodiester bond
- Exonucleases and Endonucleases (restriction enzyme)
- DNA ligase: attach 2 DNA covalently
- DNA polymerase: sDNA as template, primer, radio labeling, PCR, DNA sequencing
- Reverse transcriptase: RNA as template, primer, used for making sDNA library
Restriction enzymes
Know probability!!!
- Bacterial enzyme that cuts ds DNA in specific sequence
- Restrict entry of foreign DNA by cleaving at recognition site that’s not in host bacterium
- 4-8 bp normal length of restriction site
- Cut asymmetrically to leave with sticky ends (better) or blunt ends
What is DNA cloning
Process of making genetically identical organism thru nonsexual means. Allows specific DNA sequences to be separated from genome and placed in vector. Use bacteria to amplify DNA segments. PCR amplifies specific regions of DNA.
What are the steps of PCR?
- Denaturation at 95C (separation of ds DNA)
- Annealing at 55C: Cooled and allow 2 single stranded DNA primers to anneal to sample DNA
- DNA Chain extension at 72C: heat stable DNA polymerase extends primers attached to target DNA
- REPEAT CYCLE 30-40 times
What are the applications of PCR?
Identify single nucleotide polymorphisms
DNA/RNA quantifications
Forensic analysis
What are some methods used for DNA analysis?
Gel separation: electrophoresis
Nucleic acid hybridization: Southern blotting
DNA sequencing
Dectection of sequence polymorphisms
What is nucleic acid hybridization?
Used to ID specific DNA seq probes denatured and annealed to sample DNA that has been denatured usually labeled with radio isotope 32P
What is southern blotting?
DNA probes ID-specific DNA with complementary bases
What is Northern blotting?
DNA detects specific RNA sequence
What is Western blotting?
Use protein antibodies to detect antigens (protein-protein)
What is Southwestern blotting/
short target DNA sequence labeled and serves as probes for hybridized rxn
What are 2 examples of detection of sequence polymorphisms?
RFLP restriction fragment length polymorphism (specific)
VNTR variable # tandem repeat
Describe how to the mutation of sickle cell anemi and how is it detected?
RFLP
1 point mutation (Glu->val) so A->T at position 6 in beta globin protein chain. Becomes hydrophobic. Mutant becomes insoluble in O2 free form, crystallized, form sickle shape->anemia, thrombotic vaso-occlusion, and hemolysis.
How does the mutation in sickle cell anemia affect fragmentation?
Mutation causes loss of restriction enzyme site for MSII.
Normal DNA has 2 fragments when cut with MSII.
Mutant DNA only has 1 fragment when cut with MSII.
Huntington’s diseased is caused by what? What method would you use to find it?
VNTR
HD gene on ch. 4. Fatal neurological disorder. CAG repeat normal:<28. PreMutant: 29-39 times. Mutant >40. produce huntingtin protein causing ‘polar zipper’.
Can use Hind3 restriction enzyme to establish # of CAG repeats
What are the 2 types of DNA library and explain them
Genomic: contains DNA fragments representing entire genome of organism (exons + introns)
cDNA: contains only complementary DNA made from mRNA (exons only)
DNA vaccines
usually consist of single/multiple genes of immunogenic proteins inserted into commercially available DNA expression plasmid
Better to use heat-killed than attenuated
