Exam 1 Flashcards
What is a nucleoside?
A base and a sugar – NO phosphate
What is a nucleotide?
A base, a sugar, and a phosphate group
What is the name of the bond which attaches two nucleotides together?
Phosphodiester bond
–> Forms between the 5’ end of one sugar and the 3’ end of the next sugar
What is the name of the bond which holds together two phosphate groups?
Phosphoanhydride bonds
True or false: Phosphoanhydride bonds are not easily hydrolyzed
FALSE: These bonds ARE easily hydrolyzed. When the bond is hydrolyzed, energy is released
What three stabilizing forces are present in B DNA?
i) Placement on opposite sides of the double helix (on outside of helix, as opposed to inside)
ii) The negative charge on the phosphates are neutralized by positive charges of lysine and arginine residues of histone proteins
iii) The stacking of the aromatic rings of the bases that permits van der Waal interactions between the electron clouds that sandwich the rings
What is the bond between the base and the sugar in a nucleoside?
N-glycosidic bond
How does A form differ from B form? What type of DNA or RNA takes on the A form?
- Wider and more compact than B
- The structure assumed by dsRNA (double-stranded RNA)
How does the Z form differ from the B form? What type of DNA or RNA takes on the Z form?
- It is left-handed as opposed to right-handed
- Doesn’t really exist in nature
Describe 3-stranded DNA
-Occurs through Hoogsteen base pairing (H-binding with N’s in 5-membered (rather than 6-membered) ring from purine)
What is 4-stranded DNA? Where does it occur?
- Called G quartets or G-quadruplexes
- Occurs in G-rich areas of DNA or RNA
- Stack on top of each other in 4G coils
- Occurs often in telomeres
What is ssDNA? Where would you find it?
- It is circular DNA (bound by a phosphodiester bond from the 3’ to 5’ end)– Also called a plasmid
- Found in bacteria and archaea
Which part of a helix-turn-helix motif can fit into the major groove of DNA?
The a-helix
What part of a zinc finger motif can fit into the major groove of DNA?
The a-helix
How do DNA binding proteins work?
They recognize a specific sequence of DNA and can bind in the major groove
True or false: B-sheets can fit into the major groove of DNA
TRUE - B-sheets, along with a-helices, can both fit into the major helix of DNA in order to interact with it
How does a protein attach to DNA when interacting with it?
Through hydrogen bonds on the amino acid side chains of the protein and the phosphate backbone of the DNA (in the major groove)
Definition: Multivalency
When a lot of weak bonds all work together and the resulting bond of the two molecules is very strong (think of velcro– only one fiber of velcro is weak, but the more you have, the better the two objects stick together)
What is the molecular basis by which proteins read DNA’s encoded information? (3 answers)
i) Hydrogen Bonding
ii) Multivalent interactions
iii) Intercalation
What does a restriction enzyme do?
Restriction enzymes chop up DNA
What does an intercalating dye do? What is an example of an intercalating dye?
- And intercalating dye stains DNA so that it can be seen under UV light (typically in a gel electrophoresis experiment)
- Ethidium bromide is an intercalating dye
What is hybridization?
When a set of bases pairs with a complementary set of bases (making hydrogen bonds)
–> When one strand of DNA binds to a new strand of DNA (not when a new strand is being formed from a template strand)
Why do higher temperatures create the more accurate hybridizations?
Only perfect matches will form at higher temps because they are the only ones strong enough to resist the heat– weaker (imperfect) matches will be unable to stay bonded at high temps
What is a Western Blot used for?
To detect proteins
What is a Northern Blot used for?
To detect RNA
What is a Southern Blot used for?
To detect DNA
Definition: DNA sequencing
The technology to determine the order of base pairs along DNA molecules
–> Often requires cloning
When cloning via plasmids, why must you use the same restriction enzymes for both the plasmid and the DNA?
You need to use the same restriction enzymes for both so that they have matching sticky ends. The sticky ends will improve binding.
What are the two types of “Libraries”? What is the difference between them?
i) Genome Library
- -Contains all genes in a genome, both coding and non-coding
ii) cDNA Library
- -Contains ONLY genes that code for proteins (exons)– no introns or non-coding areas are included
Describe blue-white colony selection using the LacZ gene
- Plasmids are made that should disrupt the LacZ gene when inserted into the bacterial genome
- After mixing plasmids and bacteria, the bacteria are grown on a plate that contains a blue galactosidase compound
- Those bacteria that grow into a blue colony can still process galactosidase (because their LacZ gene is still intact), and therefore these are UNDESIRABLE colonies (do not contain the desirable plasmid)
- Colonies that grow as a white color have a disrupted LacZ gene, therefore these colonies are selected FOR, because they have the desirable plasmid
What is an oligo dT? What is its purpose?
Basically a ‘poly-T’ situation– bunch of Ts in a row (can bond to a poly-A tail)
–> Used as a universal primer for cDNA cloning
True or false: You can create tissue-specific cDNA libraries
TRUE: Different genes are expressed in different tissues, and in creating a cDNA library, only the genes that are expressed in a given cell are reverse transcribed into cDNA
What is a restriction enzyme? What do scientists use them for?
- Restriction enzymes are used by other organisms to degrade foreign DNA (a type of immune system)
- We (scientists) use them to break up long DNA molecules into manageable-sized pieces
What does an endonuclease do? What does an exonuclease do?
Endonucleases hydrolyze DNA in the interior of a double helix
-Exonucleases hydrolyze DNA from its ends
What type of endonucleases do we use to break apart DNA molecules?
Deoxyriboendonucleases
True or false: You can use PCR on DNA and proteins
FALSE: You can ONLY use PCR for DNA (also RNA). Proteins cannot be PCR’ed because during the heating step of PCR, the proteins will denature
How do you selectively amplify a gene in PCR?
By adding in primers for those specific sections of DNA or RNA
What component properties are needed in a vector?
i) Selection Marker Genes (like the lacZ gene or a drug resistant gene)
ii) Origin of Replication (NOT all vectors require a promoter, but a typical vector does require one)
iii) Restriction Site (place where the genome gets chopped in order to insert the new stuff)
What does BAC stand for?
Bacterial Artificial Chromosomes
What is an MCS? What does it stand for?
- Stands for Multiple Cloning Site
- An artificial sequence that contains the recognition sequence for different restriction enzymes
Explain Dideoxy Sequencing (also called Sanger Sequencing)
Shortest strands go through first, we can see what color is at the truncated end. So it would be like 1 nucleotide long, 2 nucleotide long, 3 nucleotide long, etc. and based on what colors come through one after the other you can see what the sequence is
What is a CONTIG?
A contiguous overlapping sequence
–Assembling a bunch of CONTIGs is what you must do if you don’t know anything about your sequence
Describe Illumina Sequencing
- Break up DNA into small pieces (~100 bp)
- Attach adapters to both ends
- Denature and attach both ends to an optical plate
- Make DNA double-stranded with DNA polymerase
- Denature to make single-stranded
- Amplify
- Carry out DNA sequencing with primers, DNA polymerase and fluorescent nucleotides
- Reads often do not go for more than 25 nucleotides
What is SKY?
Getting an entire chromosome labeled with a specific color (each chromosome having a different color to easily differentiate)
- -Whole genome FISH
- -Can reveal genetic abnormalities
Approximately what percentage of the human genome codes for proteins?
1.5%
–About 25% of made up of genes, but most of this is introns which get removed before translation
What is the start codon (starts TRANSLATION)?
AUG – Codes for methionine
–> This is also the only methionine codon, so it gets used not only as the initiator codon but also to code all methionines in proteins
What are the three stop codons (terminates TRANSLATION)?
UAA, UAG, UGA
–Do not code for anything else except stopping translation
When do introns get removed from the genome?
Introns are removed as pre-mRNA is processed to mRNA
What is spacer DNA?
DNA between genes that can be unique or repetitive
True or False: DNA Transposons are the primary transposon type in humans
FALSE: DNA transposons are called ‘fossilized’ transposons because they exist in our genome, but are no longer functional
The primary type of transposon in human genomes is nonretroviral transposons
How do nonretroviral transposons increase the length of the genome?
The receiving end has little overhangs that the transposon binds to. These overhangs get duplicated when the transposon is inserted, and this lengthens the genome
True or False: Retrotransposons use RNA to move genetic material around
TRUE - Use of reverse transcriptase
What does LINE stand for? SINE?
LINE: long interspersed nuclear elements
SINE: short interspersed nuclear elements
Aside from length, what is the major difference between LINEs and SINEs?
LINEs are autonomous, SINEs are not
–> Autonomous means that the sequence encodes its own RNA binding protein and reverse transcriptase (called L1 in humans– SINEs don’t make their own, depend upon LINEs creating enough L1 for both)