Chapter 9, Problem 1P

(a)

Summary Introduction

To draw: The structure of each end of a linear DNA fragment produced by an EcoRI restriction digest.

Introduction:

EcoRI is a restriction endonuclease enzyme isolated from species E. coli. The restriction site of EcoRI consists of six palindromic nucleotide sequences and the restriction digestion produce sticky ends.

Explanation of Solution

Explanation:

Recognition sequence of EcoRI is GAATTC.

So, the fragments produced at both the ends are:

$\begin{array}{l}\text{<msup>5<mo>′</mo></msup>-G <msup>3<mo>′</mo></msup>}\\ \text{<msup>3<mo>′</mo></msup>-CTTAA-<msup>5<mo>′</mo></msup>}\end{array}$ And $\begin{array}{l}\text{<msup>5<mo>′</mo></msup>-AATTC-<msup>3<mo>′</mo></msup>}\hfill \\ \text{<msup>3<mo>′</mo></msup>-TTAAG-<msup>5<mo>′</mo></msup>}\hfill \end{array}$

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(b)

Summary Introduction

To draw: The structure resulting from the reaction of this end sequence with DNA polymerase I and the four deoxynucleoside triphosphates.

Introduction:

DNA polymerase I is an important enzyme used in the replication of DNA. It binds to specific sequence called initiator sequence. DNA polymerase I has multiple functions which include nick translation during DNA repairing, proofreading and DNA dependent DNA polymerase activity.

Explanation of Solution

Explanation:

The strands formed after DNA polymerase I activity are:

$\begin{array}{l}\text{<msup>5<mo>′</mo></msup>-GAATT-<msup>3<mo>′</mo></msup>}\hfill \\ \text{<msup>3<mo>′</mo></msup>-CTTAA-<msup>5<mo>′</mo></msup>}\hfill \end{array}$ and $\begin{array}{l}\text{<msup>5<mo>′</mo></msup>-AATTC-<msup>3<mo>′</mo></msup>}\hfill \\ \text{<msup>3<mo>′</mo></msup>-TTAAG-<msup>5<mo>′</mo></msup>}\hfill \end{array}$

DNA polymerase enzyme adds the complementary nucleotide base pairs to the sequence, which makes the sticky end turn into blunt ends.

(c)

Summary Introduction

To draw: The sequence produced at the junction that arises if two ends with the structure derived in (b) are ligated.

Introduction:

Ligation is the process of joining two or more sequences with the help of an enzyme called DNA ligases. DNA ligase forms phosphodiester bonds between the nucleotides.

Explanation of Solution

Explanation:

The sequence obtained after ligation of the two ends is:

$\begin{array}{l}\text{<msup>5<mo>′</mo></msup>-GAATTAATTC-<msup>3<mo>′</mo></msup>}\hfill \\ \text{<msup>3<mo>′</mo></msup>-CTTAATTAAG-<msup>5<mo>′</mo></msup>}\hfill \end{array}$

DNA ligase forms the phosphodiester bonds between two nucleotides which end up joining two DNA fragments.

(d)

Summary Introduction

To draw: The structure produced if the structure derived in (a) is treated with a nuclease that degrades only single stranded DNA.

Introduction:

Nuclease enzyme is important in breaking the phosphodiester bond between the nucleotide. There are two types of nuclease enzymes endonuclease and exonuclease. Exonuclease attacks the DNA from the terminals and endonuclease attacks the DNA from anywhere between the DNA.

Explanation of Solution

Explanation:

The sequence obtained after treatment with nuclease that degrades only single stranded DNA is:

$\begin{array}{l}\text{<msup>5<mo>′</mo></msup>-G-<msup>3<mo>′</mo></msup>}\\ \text{<msup>3<mo>′</mo></msup>-C-<msup>5<mo>′</mo></msup>}\end{array}$

And

$\begin{array}{l}\text{<msup>5<mo>′</mo></msup>-C-<msup>3<mo>′</mo></msup>}\hfill \\ \text{<msup>3<mo>′</mo></msup>-G-<msup>5<mo>′</mo></msup>}\hfill \end{array}$

Nuclease enzyme removed the nucleotide sequences present at the end turning the sticky ends into blunt ends.

(e)

Summary Introduction

To draw: The sequence of the junction produced if an end with structure (b) is ligated to an end with structure (d).

Introduction:

Ligation is the process of joining two or more sequences with the help of an enzyme called DNA ligases. DNA ligase forms phosphodiester bonds between the nucleotides.

Explanation of Solution

Explanation:

The resulting sequence obtained after ligation structure (b) with structure (d) is:

$\begin{array}{l}\text{<msup>5<mo>′</mo></msup>-GAATTC-<msup>3<mo>′</mo></msup>}\hfill \\ \text{<msup>3<mo>′</mo></msup>-CTTAAG-<msup>5<mo>′</mo></msup>}\hfill \end{array}$

The ligase enzyme joined the ends of the two separate DNA fragments and formed a continuous sequence.

(f)

Summary Introduction

To draw: The structure of the end of a linear DNA fragment that was produced by a PvuII restriction digest.

Introduction:

PvuII restriction enzyme is isolated from a bacterium known as Proteus vulgaris. The recognition sequence of PvuII is made up of six nucleotide sequence and the ends produced are blunt ends

Explanation of Solution

Explanation:

The sequences obtained after digestion with restriction enzyme PvuII are:

$\begin{array}{l}\text{<msup>5<mo>′</mo></msup>-CAG-<msup>3<mo>′</mo></msup>}\\ \text{<msup>3<mo>′</mo></msup>-GTC-<msup>5<mo>′</mo></msup>}\end{array}$ and $\begin{array}{l}{\text{5}}^{\prime }-CTG-{\text{3}}^{\prime }\\ {\text{3}}^{\prime }-GAC-{\text{5}}^{\prime }\end{array}$

PvuIIproduces blunt ends on digesting the DNA fragment and the recognition sequence for PvuII is 5ʹ CAGCTG 3ʹ                              PvuII is  CAGCTG $3^{\prime }$

(g)

Summary Introduction

To determine: The sequence of the junction produced if an end with structure (b) is ligated to an end with structure (f).

Introduction:

Ligation is the process of joining two or more sequences with the help of an enzyme called DNA ligases. This enzyme is important in repairing single stranded breaks in the DNA. DNA ligase forms phosphodiester bonds between the nucleotides.

Explanation of Solution

Explanation:

The resulting sequence obtained after ligation structure (b) with structure (f) is:

$\begin{array}{l}{\text{5}}^{\prime }-CAGAATTC-{\text{3}}^{\prime }\hfill \\ {\text{3}}^{\prime }-GTCTTAAG-{\text{5}}^{\prime }\hfill \end{array}$

The ligase enzyme joined the ends of the two separate DNA fragments by catalyzing the formation of phosphodiester bonds between them.

(h)

Summary Introduction

To determine: A protocol for removing a EcoRI restriction site from DNA and incorporate BamHI restriction site at the same location.

Introduction:

BamHI restriction enzyme is isolated from a bacterium known as Bacillus amyloliquefaciens. BamHI is a type II restriction enzyme and the recognition sequence is made up of six nucleotide sequence and the ends produced are four nucleotide long sticky ends.

Explanation of Solution

Explanation:

First, the DNA is digested with EcoRI. It generates staggered ends these ends are treated with polymerase I enzyme and four nucleotide base pair. DNA polymerase binds the NTPs to the complementary sequence of the DNA, and converts sticky ends to blunt end.

After conversion of sticky ends produced by EcoRI into blunt ends the DNA fragment is ligated with a synthetic fragment which has recognition sequence for BamHI enzyme this will result into conversion of EcoRI restriction site into BamHI.

The sticky ends produced by restriction digestion can be converted to blunt ends by using polymerase enzyme and the four nucleotide tri phosphate containing solution. Similarly a restriction site can be added by ligation with the ligase enzyme and synthetic nucleotide sequence.

(i)

Summary Introduction

To design: Four different short synthetic double-stranded DNA fragments that would permit ligation of structure (a) with a DNA fragment produced by a PstI restriction digest.

Introduction:

EcoRI is a restriction endonuclease enzyme isolated from species E. coli. The restriction site of EcoRI consists of six palindromic nucleotide sequences and the restriction digestion produce sticky ends. PstI is a restriction endonuclease enzyme isolated from species Providencia stuartii. It is a type II restriction enzyme and produces sticky ends.

Explanation of Solution

Explanation:

The sequence in which the final junction contains the recognition sequences for both EcoRI and PstI is:

$\begin{array}{l}\text{<msup>5<mo>′</mo></msup>AATTCNNNCTGCA-<msup>3<mo>′</mo></msup>}\hfill \\ {\text{3}}^{\prime }\text{GNNNG}{\text{5}}^{\prime }\hfill \end{array}$

The sequence that contains only the EcoRI restriction segment is:

$\begin{array}{l}\text{<msup>5<mo>′</mo></msup>AATTCNNNGTGCT-<msup>3<mo>′</mo></msup>}\\ {\text{3}}^{\prime }\text{GNNNC}{\text{5}}^{\prime }\end{array}$

The sequence that contains the recognition site for only PstI is:

$\begin{array}{l}\text{<msup>5<mo>′</mo></msup>AATTGNNNCTGCA-<msup>3<mo>′</mo></msup>}\hfill \\ {\text{3}}^{\prime }\text{CNNNG}{\text{5}}^{\prime }\hfill \end{array}$

The sequence that has neither of the two recognition sites present is:

$\begin{array}{l}\text{<msup>5<mo>′</mo></msup>ATTTCNNNCTTCA-<msup>3<mo>′</mo></msup>}\hfill \\ {\text{3}}^{\prime }\text{GNNNG}{\text{5}}^{\prime }\hfill \end{array}$

The recognition site for specific enzyme is specifically based on the nucleotide sequence. The enzyme binds to a specific nucleotide sequence present inside the DNA and cleaves it producing sticky or blunt ends.