ASCP MOLECULAR BIOLOGY CERTIFICATION EXAM
Pyrimidine - answer- One carbon ring
Cytosine, Thymine, Uracil
Purine - answer- Two carbon rings
Adenine, Guanine
How are nucleotides joined together? - answer- Condensation to form phosphodiester
bond
What is the function of mRNA? - answer- Carries genetic info out of nucleus
Transcript translated to protein
What is the function of tRNA? - answer- Carries aa to ribosome
Anticodon pairs with codon on mRNA strand
What is the function of rRNA? - answer- part of ribosome structure
most abundant RNA
coordinated coupling of tRNA to mRNA codons
Feedback inhibition - answer- Product of pathway is noncompetitive inhibitor
Binds to allosteric site to slow down rxn b/c too much product
Exonucleases - answer- Degrades nucleic acids by removing one terminal nt at a time
Cleaves phosphodiester bond at end of chain
5' --> 3' and 3' --> 5'
Endonucleases (Prok) - answer- Restriction enzymes
Cleaves phoshpodiester bonds w/i poly-nt chain
Recognition site is palindromic sequence
Types I-V
ORI sites - answer- nt sequence where replication is initiated
Topoisomerase I - answer- Induces ss breaks
Remove DNA supercoils during TXN and DNA replication; for strand breakage during
recombination; for chr condensation; and to disentangle intertwined DNA during mitosis
topoisomerase II - answer- cuts both strands of one DNA double helix, passes another
unbroken DNA helix through it, and then reanneals the cut strands
Gyrase (topoisomerase II) - answer- Unwinds supercoiling caused by unwinding at the
rep fork by introducing DSBs
Helicase - answer- Breaks H-bonds of double helix at the replication fork
,Primase - answer- DNApol α (DNA dep RNA pol)
adds short segments of complementary
RNA to ssDNA template (primers), serves as starting points for replication
single-strand DNA binding proteins (SSBPs) - answer- Binds ssDNA and prevents it
from re-annealing during TXN, replication, repair, and recombination
Okazaki fragments - answer- Short fragments of DNA synthesized by DNApol δ using
the lagging strand (3'->5') as a template
Ligase - answer- Closes gaps in DNA
Catalyzes phosphodiester bond between 3'OH and 5'P
What are the steps in DNA replication? - answer- 1. Initiate
2. Elongate
3. Terminate
Telomeres - answer- Repeat sequence (TTAGGG) at the ends of chr, protect chr from
degradation
RNA polymerase - answer- DNA dependent RNApol
Transcribes DNA template to RNA (3'-->5'; anti-parallel)
Splicesomes - answer- Complex of snRNPs
Removes introns from pre-mRNA and splices exons together
Enhancers - answer- Short regions of DNA that bind proteins (TXN factors) that
enhance TXN of a gene
Poly-A tail - answer- Prevents mRNA from being degraded in cytoplasm
100-250 A's at 3' end
5' cap - answer- 5'-5' pyrophosphate bridge to a methylated G added to 5' end of a
mRNA
Protects against degradation and as a recognition signal for TLN apparatus
aminoacyl tRNA - answer- tRNAs that carry amino acids
Ribosomes - answer- Where TLN occurs
Prok: 30s and 50s
Euk: 40s and 60s
Catalyzes peptide bond between a.a.'s
What is the path of a tRNA in a ribosome? - answer- Acceptor > Peptidyl > Exit
, How is translation initiated? - answer- small rRNA (40S) subunit binds mRNA and scans
for start codon (AUG)
Met-tRNA is brought to the P site
Large rRNA (60S) subunit binds
Microarray steps - answer- 1. isolate mRNA from cells
2. RT to get labeled cDNA copies of mRNA
3. cDNA washed over slide. cDNA sticks to comp sequence
4. use laser to read fluorescent tags
Southern Blot - answer- Detect a large DNA fragment among many
Target: DNA, probe: DNA
What can Southern Blots be used to detect? - answer- Deletions/insertions
Point mutations
Polymorphisms
Structural rearrangements
Southern Blot steps - answer- 1. RE digest to fragment DNA
2. Run on gel to separate
3. Soak gel in alkali/NaOH to denature dsDNA
4. Transfer ssDNA fragments to positively charged membrane (blot)
5. Fix to filter by heat (80C) or UV crosslink
6. Incubate (hybridize) blot w/ radioactively labeled ssDNA comp probe
7. Autoradiograph
Heterduplex Analysis (HA) - answer- use for known gene, unknown mutation
mutation screening
bands on gel --> retarded migration from WT due to seq differences
Heterduplex Analysis steps - answer- 1. PCR
2. Mix sample and CTR DNA together
3. Denature PCR using heat
4. Cool slowly to rt
5. Add denaturing loading buffer
6. Run on MDE gel
Single-Stranded Conformational Polymorphism Ananlysis (SSCP) - answer- Used for
known gene, unknown mut
Mutation screening
Short PCR products form 3D conformation when cooled --> muts have different
conformation than WT
Non-denaturing PAGE, muts migrate different than WT
What makes DNA negatively charged? - answer- Phosphate groups of the
phosphate:ribose backbone
Pyrimidine - answer- One carbon ring
Cytosine, Thymine, Uracil
Purine - answer- Two carbon rings
Adenine, Guanine
How are nucleotides joined together? - answer- Condensation to form phosphodiester
bond
What is the function of mRNA? - answer- Carries genetic info out of nucleus
Transcript translated to protein
What is the function of tRNA? - answer- Carries aa to ribosome
Anticodon pairs with codon on mRNA strand
What is the function of rRNA? - answer- part of ribosome structure
most abundant RNA
coordinated coupling of tRNA to mRNA codons
Feedback inhibition - answer- Product of pathway is noncompetitive inhibitor
Binds to allosteric site to slow down rxn b/c too much product
Exonucleases - answer- Degrades nucleic acids by removing one terminal nt at a time
Cleaves phosphodiester bond at end of chain
5' --> 3' and 3' --> 5'
Endonucleases (Prok) - answer- Restriction enzymes
Cleaves phoshpodiester bonds w/i poly-nt chain
Recognition site is palindromic sequence
Types I-V
ORI sites - answer- nt sequence where replication is initiated
Topoisomerase I - answer- Induces ss breaks
Remove DNA supercoils during TXN and DNA replication; for strand breakage during
recombination; for chr condensation; and to disentangle intertwined DNA during mitosis
topoisomerase II - answer- cuts both strands of one DNA double helix, passes another
unbroken DNA helix through it, and then reanneals the cut strands
Gyrase (topoisomerase II) - answer- Unwinds supercoiling caused by unwinding at the
rep fork by introducing DSBs
Helicase - answer- Breaks H-bonds of double helix at the replication fork
,Primase - answer- DNApol α (DNA dep RNA pol)
adds short segments of complementary
RNA to ssDNA template (primers), serves as starting points for replication
single-strand DNA binding proteins (SSBPs) - answer- Binds ssDNA and prevents it
from re-annealing during TXN, replication, repair, and recombination
Okazaki fragments - answer- Short fragments of DNA synthesized by DNApol δ using
the lagging strand (3'->5') as a template
Ligase - answer- Closes gaps in DNA
Catalyzes phosphodiester bond between 3'OH and 5'P
What are the steps in DNA replication? - answer- 1. Initiate
2. Elongate
3. Terminate
Telomeres - answer- Repeat sequence (TTAGGG) at the ends of chr, protect chr from
degradation
RNA polymerase - answer- DNA dependent RNApol
Transcribes DNA template to RNA (3'-->5'; anti-parallel)
Splicesomes - answer- Complex of snRNPs
Removes introns from pre-mRNA and splices exons together
Enhancers - answer- Short regions of DNA that bind proteins (TXN factors) that
enhance TXN of a gene
Poly-A tail - answer- Prevents mRNA from being degraded in cytoplasm
100-250 A's at 3' end
5' cap - answer- 5'-5' pyrophosphate bridge to a methylated G added to 5' end of a
mRNA
Protects against degradation and as a recognition signal for TLN apparatus
aminoacyl tRNA - answer- tRNAs that carry amino acids
Ribosomes - answer- Where TLN occurs
Prok: 30s and 50s
Euk: 40s and 60s
Catalyzes peptide bond between a.a.'s
What is the path of a tRNA in a ribosome? - answer- Acceptor > Peptidyl > Exit
, How is translation initiated? - answer- small rRNA (40S) subunit binds mRNA and scans
for start codon (AUG)
Met-tRNA is brought to the P site
Large rRNA (60S) subunit binds
Microarray steps - answer- 1. isolate mRNA from cells
2. RT to get labeled cDNA copies of mRNA
3. cDNA washed over slide. cDNA sticks to comp sequence
4. use laser to read fluorescent tags
Southern Blot - answer- Detect a large DNA fragment among many
Target: DNA, probe: DNA
What can Southern Blots be used to detect? - answer- Deletions/insertions
Point mutations
Polymorphisms
Structural rearrangements
Southern Blot steps - answer- 1. RE digest to fragment DNA
2. Run on gel to separate
3. Soak gel in alkali/NaOH to denature dsDNA
4. Transfer ssDNA fragments to positively charged membrane (blot)
5. Fix to filter by heat (80C) or UV crosslink
6. Incubate (hybridize) blot w/ radioactively labeled ssDNA comp probe
7. Autoradiograph
Heterduplex Analysis (HA) - answer- use for known gene, unknown mutation
mutation screening
bands on gel --> retarded migration from WT due to seq differences
Heterduplex Analysis steps - answer- 1. PCR
2. Mix sample and CTR DNA together
3. Denature PCR using heat
4. Cool slowly to rt
5. Add denaturing loading buffer
6. Run on MDE gel
Single-Stranded Conformational Polymorphism Ananlysis (SSCP) - answer- Used for
known gene, unknown mut
Mutation screening
Short PCR products form 3D conformation when cooled --> muts have different
conformation than WT
Non-denaturing PAGE, muts migrate different than WT
What makes DNA negatively charged? - answer- Phosphate groups of the
phosphate:ribose backbone
