BIOC441 Week 8

TA: Julia Joo

 Techniques
• UV light absorbance
• Tm, percent composition of base types
• PCR
• Restriction enzymes, Gibson assembly
• Deep/Illumina sequencing
• Mapping histones, large stretches of DNA
• Sanger sequencing
• Good for gene sequencing
Recombinant
Plasmid DNA
plasmid
ligate
RE digest

insert DNA
Experiments
• Chargaff’s rule
• X-ray diffraction
• Dickerson’s crystal
• Alternative bases
• Ultracentrifugation of different levels of supercoiling
• Gels after exposure to topoisomerases
• Optical trap
• Meselson-Stahl
 DNA polymerase

PPi

• Fairly accurate; error rate is ~1/105 bases

• More likely to make errors in presence of?
• Mg2+ stabilizes
• Negative phosphate groups
• Deprotonated 3’ oxygen on
nascent strand
 DNA polymerase

PPi

• Fairly accurate; error rate is ~1/105 bases

• More likely to make errors in presence of
• Mg2+ stabilizes • Alkylating agents
• Negative phosphate groups • Oxidative damage
• Deprotonated 3’ oxygen on • Deamination of 5-methyl-C
nascent strand
You are setting up a plasmid assembly using EcoRI. Your plasmid
backbone is 2kb and your insert is 3kb. After transforming into E.coli,
you run a gel of your plasmid to check your assembly. To your dismay,
you see the following gel. What should your gel have looked like? What
was wrong with your assembly?
 You are setting up a plasmid assembly using EcoRI. Your plasmid
backbone is 2kb and your insert is 3kb. After transforming into E.coli,
you run a gel of your plasmid to check your assembly. To your dismay,
you see the following gel. What should your gel have looked like? What
was wrong with your assembly? Expected

There is another EcoRI site within the insert, cutting it

into 2kb and 1kb. Both pieces are able to combine
with the plasmid at the EcoRI sites.

Digest, ligate

EcoRI sites

EcoRI site
2kb 3kb Expected, 5kb 4kb 3kb
You are setting up a plasmid assembly using EcoRI. Your plasmid
backbone is 2kb and your insert is 3kb. After transforming into E.coli,
you run a gel of your plasmid to check your assembly. To your dismay,
you see the following gel. What should your gel have looked like? What
was wrong with your assembly? Expected

If you sequenced the mixture of plasmids you got in

your assembly, what would the Sanger sequencing
map results look like? Draw a rough sketch.
You are setting up a plasmid assembly using EcoRI. Your plasmid
backbone is 2kb and your insert is 3kb. After transforming into E.coli,
you run a gel of your plasmid to check your assembly. To your dismay,
you see the following gel. What should your gel have looked like? What
was wrong with your assembly? Expected

If you sequenced the mixture of plasmids you got in

your assembly, what would the Sanger sequencing
map results look like? Draw a rough sketch.
 You wish to sequence the following gene with Sanger sequencing using a 20bp
primer. You prepare 4 reaction mixtures (below) and run each reaction on it’s
own lane on an agarose gel. Draw all of the bands you would expect to see in
that reaction.

Lane: 1 2 3 4

Size (bp)
Sequence: 5’ – A T G G T G A C C G - 3’ 30
29
Tube 1: dATP, dCTP, dGTP, dTTP, ddATP 28
Tube 2: dATP, dCTP, dGTP, dTTP, ddTTP 27
Tube 3: dATP, dCTP, dGTP, dTTP, ddGTP 26
Tube 4: dATP, dCTP, dGTP, dTTP, ddCTP 25
24
All tubes contain a polymerase, Mg2+, buffer, and the primer. 23
22
21
 You wish to sequence the following gene with Sanger sequencing using a 20bp
primer. You prepare 4 reaction mixtures (below) and run each reaction on it’s
own lane on an agarose gel. Draw all of the bands you would expect to see in
that reaction.

A T G C
Lane: 1 2 3 4

Size (bp)
Sequence: 5’ – A T G G T G A C C G - 3’ 30
29
Tube 1: dATP, dCTP, dGTP, dTTP, ddATP 28
Tube 2: dATP, dCTP, dGTP, dTTP, ddTTP 27
Tube 3: dATP, dCTP, dGTP, dTTP, ddGTP 26
Tube 4: dATP, dCTP, dGTP, dTTP, ddCTP 25
24
All tubes contain a polymerase, Mg2+, buffer, and the primer. 23
22
21
Name the following molecules. Circle the N atoms that will be labeled first with
15N, and briefly explain why.
Name the following molecules. Circle the N atoms that will be labeled first with
15N, and briefly explain why.

In orotidylate (left), this N comes from carbamoyle

phosphate, which is produced from NH3. For IMP (right),
these N come from glutamine, the initial ammonia carrier.
The backbone of DNA is more than 100 times as stable as the backbone
of RNA. Explain why RNA is less stable than DNA.
The backbone of DNA is more than 100 times as stable as the backbone
of RNA. Explain why RNA is less stable than DNA.

The 2´–OH in RNA can attack the phosphodiester bond formed at the
adjacent 3´ position, resulting in cleavage of the backbone and
formation of a 2´,3´cyclic monophosphate product. The cyclic
product spontaneously rearranges to a 2´ or 3´ monophosphate.
In a spontaneous non-enzymatic reaction, water attacks deoxycytidine
triphosphate (dCTP), resulting in a deaminated product. What does this fact
suggest about the presence of deoxythymidine (dT) in all modern DNA
genomes?
In a spontaneous non-enzymatic reaction, water attacks deoxycytidine
triphosphate (dCTP), resulting in a deaminated product. What does this fact
suggest about the presence of deoxythymidine (dT) in all modern DNA
genomes?

Deaminated cytosine = uracil. Thus, if dU were a normal base in DNA, the cell could not
discriminate between correctly-inserted dU and daminated dC. However, in DNA dU is not
used and dT is. dUTPase can distinguish between dUTP and dTTP, thereby lowering the
likelihood of mutation due to this spontaneous chemical reaction. Together these
considerations help to explain a deep and ancient question: why DNA uses T while RNA
seems to get by with U.