L7-Genetic Engineering

Question 1

Define a restriction enzyme and why they are useful to a molecular biologist?

Answer 1

A restriction enzyme recognizes specific base sequences (recognition sequences) within DNA and cut the DNA. They are essential for inserting sequences into vectors by making sticky ends.

Question 2

How do nucleic acids separate using electrophoresis?

Answer 2

• -Samples are put in an agarose gel which hinders molecular movement by size
• -DNA samples move towards anode/positive electrode.
• -Gels are stained with a compound such as ethidium bromide fluoresce under UV light.

Question 3

How can you use restriction enzymes to classify microorganisms?

Answer 3

• -Purified DNA can be cut by a restriction enzyme.
• -Fragment size is determined comparison with a standard sample to create a restriction map.
• -Restriction map can be used to classify organisms with related DNA sequences.

Question 4

Explain the difference between a Northern and Southern Blot.

Answer 4

Both require probes (of known sequence) to bind to target DNA fragments separated by electrophoresis. After separation, they are transferred to a synthetic membrane.

Northern-RNA is the target sequence and DNA or RNA is the probe.
Southern-DNA is the target and DNA or RNA is the probe.

Question 5

Explain the steps of molecular cloning.

Answer 5

Molecular cloning-A fragment of DNA is isolated and replicated.

• -Isolation and fragmentation of source DNA (total genomic DNA, synthesized from RNA, gene from PCR or synthetic DNA made in vitro) cut by a restriction enzyme.
• -Inserting the DNA fragment into cloning vector by use of DNA ligase.
• -Introduction of cloned DNA into a host organism.

Question 6

Describe how you screen clones to find the one you are interested in.

Answer 6

• -look for a protein produced by the gene
• -select a marker such as antibiotic resistance so only the colonies with the gene in question survive
• -look for the colonies that regain wild-type phenotype if it is a defective mutant gene
• -most common-DNA sequencing or restriction digests on plasmids extracted from a large number of colonies

Question 7

How is site-directed mutagenesis useful for enzymologists?

Answer 7

It allows for a change to any base pair in a specific gene and to link virtually any aspect of an enzyme’s activity to the change in base pair.

Question 8

Explain the difference between reporter genes and gene fusions.

Answer 8

Reporter Gene—encodes a protein that is easy to detect and assay

Gene fusions-segments from two different genes. Ex. Promoter with a reporter gene

Question 9

Why are gene fusions useful for studying gene regulation?

Answer 9

If measuring the levels of the natural gene product is difficult or time consuming, a gene fusion allows for the assay of the product of the reporter gene to assess how the gene is regulated.

Question 10

What is the difference between a BAC and YAC?

Answer 10

YACs (yeast artificial chromosome) can hold more DNA than BACs (bacterial artificial chromosome). There is a greater problem with recombination and rearrangement of the cloned DNA within yeast than with E. coli and thus BACs are now more widely used in genomic cloning than YACs.

Question 11

Why are M13 cloning vectors useful for sequencing foreign DNA?

Answer 11

It is easy to insert DNA into M13 (which can provide a continuous source of phage DNA). Since the base sequence near the cut site is known, it is possible to construct a oligonucleotide primer complenetary to this region and use this to determine the sequence of the DNA downstream from the point.