Lecture 15 - Gene Cloning

What is step 1 for DNA cloning?

1. Digest E.coli DNA with a R.E (e.g Hind III). You have to separate the fragment from all other fragments, by ligating it with a cloning vector.

What is steps for obtaining a cloning vector?

1. Digest vector with HindIII. 2. Digest vector with alkaline phosphatase. This prevents self-ligation. 3. Mix genomic digest and vector with DNA ligase.

What are the possible ligation products?

1. Self-ligated genomic DNA. 2. Self-ligated plasmid DNA. 3. Recombinant plasmid DNA. All transform E.coli cells with the ligated DNA, and select ampicillin resistant colonies.

What is a di-deoxynucleotide?

A deoxynucleotide analogue that lack at 3'-OH.

What is a cloning vector?

An autonomously replicating DNA molecule that reproduces avidly within its host.

What are the other types of Cloning Vectors?

Bacteriophage: - Based on phage λ - Can clone up to 20kb by replacing lysogenic functions - Can be packaged in phage heads for efficient transfection. Cosmids: - A plasmid containing a phage λ "cos" site enabling packaging into phage heads. - Can clone up to 40kb DNA - Multicopy, but some inserts unstable Fosmids: - A cosmid with the origin of replication from the F- factor - Can clone up to 40kb DNA - Single-copy, so inserts much more stable BACs: - Bacterial Artificial Chromosomes - Can clone up to 300 kb DNA - Low copy.

What is an alternative approach to cloning DNA?

Clone DNA copied from messenger RNA 'complementary DNA' cDNA.

What is the function of DNA ligase?

DNA ligase joins DNA molecules together. DNA ligase makes a phosphodiester bond between the terminal 5'-phosphate on one DNA molecule and the terminal 3'-OH on another.

What does the DNA ligase do in cDNA synthesis?

DNA ligase ligates a linker-primer to the cDNA.

How can cDNA be made into double stranded?

DNA polymerase. The double-stranded cDNA can not be inserted to the vector.

What are Exons and Introns?

Exons are expressed sequences. Introns are intervening sequences. Exons are present in mRNA but Introns are not.

How do you identify colonies containing the beta-globin gene via colony hybridisation?

Hybridise the filter with a cloned, radioactively beta-glovin probe, and after washing, expose the filter to X-ray film. The probe will anneal with complementary sequences, identifying the colonies containing the beta-globin gene. These colonies can then be picked from the master plate and propagated.

What happens when a dideoxynucleotide is incorporated into a growing DNA chain?

It blocks further polymerisation.

Where is the phosphodiester bond made?

It is made between the 5'-phosphate of the incoming dNTP, and the free 3'-OH group of the chain being extended.

Regarding cDNA synthesis, what happens at the 3' end of eukaryotic mRNA

It is polyadenylated.

What makes a good cloning vector?

It must be small - Will have a few sites for restriction enzymes, other than those used for cloning DNA fragments. Has genetic markers for selection - this enables easy selection of transformants and recombinants. Reproduces itself avidly in its host - hundreds of copies per cell: allows large quantities of the cloned sequence to be purified.

What did Sanger win his two Nobel Prizes for?

One for sequencing proteins, the second for developing the most commonly used DNA sequencing method.

Do we still need to construct cloned libraries?

Originally, only cloned DNA could be sequenced, this is no more due to modern DNA tech, making it not always necessary. However it is still helpful for the assembly of accurate genome sequences. It is however necessary to be able to obtain clones of individual genes or cDNA for experimental applications such as targeted mutagenesis and industrial processes such as recombinant pharmaceutical protein production.

What does Reverse Transcriptase do in cDNA synthesis?

R.T copies the mRNA as a single-strand cDNA.

How much of the human genome actually encodes for proteins?

Roughly 5% encodes for prroteins. However, even then the protein coding sequences are interrupted by non-coding DNA 'introns'.

Where does the cloning site lie and what does this result if ligated?

The cloning site lies within the beta-galactosidase gene, so that ligation with a genomic fragment will result in this being disrupted. The enzyme, beta-galactosidase will turn a galactoside into an insoluble blue dye.

What principle is used in the most common method of DNA sequence determination?

The enzyme DNA polymerase will copy a template strand, by extending the 3'-OH end of an oligonucleotide primer annealed to it.

How does a polynucleotide grow?

They grow when a nucleotide are added to their 3'-ends to make a 5'->3' phosphodiester bond.