MOLECULAR BIOLOGY EXAM: LATEST A+ QUESTIONS
AND ANSWERS BREAKDOWN
Why do we have minor and major grooves in the DNA?
it result from the overall geometry of the Watson-Crick base-pair, because of the glycosidic
bond in each base-pair
Genomes differ in..
% GC
Length
Number of chromosomes
Linear or circular
Single or double strand
DNA or RNA
Complexity (amount of repetitive sequences)
number of bases/nucleotides
Chromatin
DNA+Protein
Histones' characteristics
Positively charged
Highly conserved
Very abundant
Similar structure
How dsDNA can be distinguished from ssDNA?
When the DNA is in the duplex state (dsDNA), interactions between base pairs decrease the UV
absorbance relative to that of single strands. When the DNA is in the single strand state the
interactions are much weaker, due to the decreased proximity, and the UV absorbance is higher
than that in the duplex state (Hyperchromicity).
In order to promote denaturation we can use:
Heat
Salt conc.
Basic pH
Reagents
תלות ה- Tm ביחסAT : GC
1% עליה של. היא ליניאריתGCתגרום לעלית ה- Tm 0.4° בc
,כיצד ישפיע אורך המולקולה על ה- Tm?
ה-Tm במולקולות אלו ככל.תלוי באורך המולקולה באופן לינארי רק במולקולות דנ"א קצרות
ה,שהמולקולה ארוכה יותר-Tm גבוה יותר.
How is renaturation specific?
because %C=%G and %A=%T then based renaturation is "complementary"
Nucleation
שלב.אזורים קומפלימנטריים נמשכים זה לזה ומתחילים להיווצר אזורים קצרים של סליל כפול
והוא תלוי בריכוז ה,זה הוא שלב קובע המהירות ברנטורציה- ssDNA בתמיסה.
Zippering
רץ' " ליצירת אזורים ארוכים של-'סיבי מתפשט באפקט דמויי "ריצ- האזור הדוdsDNA.
Why is DNA negatively charged?
because of the phosphate group
solve the problem of adding a primer with less than 100% identity
we can "force" it to cling to the the complenatory DNA by lowering the temperature or
increasing the salt concentration (but if the temp is lowered too much the primer will cling to
any sequence and wouldn't be specific)
how many BP around Histones?
150 bp around it, and the linker (between 2 histones) has 50 bp
DNA pol I activities:
5′-3′ polymerase, 3′-5′ exonuclease (for proofreading), and 5′-3 ′ exonuclease (for nick
translation, excision repair, and hydrolysis of the RNA primers during DNA replication).
Klenow Fragment
it is the Large Fragment of DNA Polymerase I. It exhibits 5'=>3' polymerase activity and 3'=>5'
exonuclease (proofreading) activity, but lacks 5'=>3' exonuclease activity of DNA polymerase I.
Do we need proofreading DNA in PCR?
IT DEPENDS
sometimes we want it to do proof reading (like when we are trying to clone), but this is very
expensive to buy the good enzymes
other times, we just don't care
What do we need for PCR?
, 1) Template - DNA fragment to be amplified (the strand from which a new strand is
synthesized).
2) Primers - 2 short DNA molecules whose sequence flanks the target sequence
Forward primer
Reverse primer
3) Nucleotides dATP, dCTP, dGTP, dTTP (dNTP)
4) Buffer maintains pH & contains salt (MgCl2-enzyme cofactor)
5) Taq DNA polymerase - to actually do the work
PCR reaction
1) Denaturation-
the two strands of DNA will be used as a template.
No helicase and No SSB!
2)Annealing -
primers hybridization to complementary sequences.
Primer concentration is high to prevent renaturation of the original DNA strands.
3) Elongation-
new DNA strand synthesis by DNA polymerase. The product is defined by the template strand.
how do primers look at each other?
with their eyes!
JK
only at the 3' end (they work from their 5' to 3')
Why do we use DNA primer in PCR (not RNA)?
DNA is a much more stable molecule (RNA is very unstable!), and because the hybrid between
two DNA's is better, and because we don't want to worry about replacing the RNA primer at the
end.
What is needed for replication?
...
How does high salt conc. influence denaturation kinetics
Tm is sensitive to Na+ concentration.
Na+ acts to shield the negative charges of the sugar-phosophate backbone from interacting
with one another. The repulsion between the negatively charged phosphate backbones is the
major force destabilizing the double helix, therefore increasing Na+ concentration increases
helix stability and decreasing Na+ concentration decreases helix stability.
DNA hybrid length and hybridization
AND ANSWERS BREAKDOWN
Why do we have minor and major grooves in the DNA?
it result from the overall geometry of the Watson-Crick base-pair, because of the glycosidic
bond in each base-pair
Genomes differ in..
% GC
Length
Number of chromosomes
Linear or circular
Single or double strand
DNA or RNA
Complexity (amount of repetitive sequences)
number of bases/nucleotides
Chromatin
DNA+Protein
Histones' characteristics
Positively charged
Highly conserved
Very abundant
Similar structure
How dsDNA can be distinguished from ssDNA?
When the DNA is in the duplex state (dsDNA), interactions between base pairs decrease the UV
absorbance relative to that of single strands. When the DNA is in the single strand state the
interactions are much weaker, due to the decreased proximity, and the UV absorbance is higher
than that in the duplex state (Hyperchromicity).
In order to promote denaturation we can use:
Heat
Salt conc.
Basic pH
Reagents
תלות ה- Tm ביחסAT : GC
1% עליה של. היא ליניאריתGCתגרום לעלית ה- Tm 0.4° בc
,כיצד ישפיע אורך המולקולה על ה- Tm?
ה-Tm במולקולות אלו ככל.תלוי באורך המולקולה באופן לינארי רק במולקולות דנ"א קצרות
ה,שהמולקולה ארוכה יותר-Tm גבוה יותר.
How is renaturation specific?
because %C=%G and %A=%T then based renaturation is "complementary"
Nucleation
שלב.אזורים קומפלימנטריים נמשכים זה לזה ומתחילים להיווצר אזורים קצרים של סליל כפול
והוא תלוי בריכוז ה,זה הוא שלב קובע המהירות ברנטורציה- ssDNA בתמיסה.
Zippering
רץ' " ליצירת אזורים ארוכים של-'סיבי מתפשט באפקט דמויי "ריצ- האזור הדוdsDNA.
Why is DNA negatively charged?
because of the phosphate group
solve the problem of adding a primer with less than 100% identity
we can "force" it to cling to the the complenatory DNA by lowering the temperature or
increasing the salt concentration (but if the temp is lowered too much the primer will cling to
any sequence and wouldn't be specific)
how many BP around Histones?
150 bp around it, and the linker (between 2 histones) has 50 bp
DNA pol I activities:
5′-3′ polymerase, 3′-5′ exonuclease (for proofreading), and 5′-3 ′ exonuclease (for nick
translation, excision repair, and hydrolysis of the RNA primers during DNA replication).
Klenow Fragment
it is the Large Fragment of DNA Polymerase I. It exhibits 5'=>3' polymerase activity and 3'=>5'
exonuclease (proofreading) activity, but lacks 5'=>3' exonuclease activity of DNA polymerase I.
Do we need proofreading DNA in PCR?
IT DEPENDS
sometimes we want it to do proof reading (like when we are trying to clone), but this is very
expensive to buy the good enzymes
other times, we just don't care
What do we need for PCR?
, 1) Template - DNA fragment to be amplified (the strand from which a new strand is
synthesized).
2) Primers - 2 short DNA molecules whose sequence flanks the target sequence
Forward primer
Reverse primer
3) Nucleotides dATP, dCTP, dGTP, dTTP (dNTP)
4) Buffer maintains pH & contains salt (MgCl2-enzyme cofactor)
5) Taq DNA polymerase - to actually do the work
PCR reaction
1) Denaturation-
the two strands of DNA will be used as a template.
No helicase and No SSB!
2)Annealing -
primers hybridization to complementary sequences.
Primer concentration is high to prevent renaturation of the original DNA strands.
3) Elongation-
new DNA strand synthesis by DNA polymerase. The product is defined by the template strand.
how do primers look at each other?
with their eyes!
JK
only at the 3' end (they work from their 5' to 3')
Why do we use DNA primer in PCR (not RNA)?
DNA is a much more stable molecule (RNA is very unstable!), and because the hybrid between
two DNA's is better, and because we don't want to worry about replacing the RNA primer at the
end.
What is needed for replication?
...
How does high salt conc. influence denaturation kinetics
Tm is sensitive to Na+ concentration.
Na+ acts to shield the negative charges of the sugar-phosophate backbone from interacting
with one another. The repulsion between the negatively charged phosphate backbones is the
major force destabilizing the double helix, therefore increasing Na+ concentration increases
helix stability and decreasing Na+ concentration decreases helix stability.
DNA hybrid length and hybridization
