Chapter 41 - Recombinant DNA Techniques

What is RNA interference (RNAi)?

Uses double-stranded RNA to target and degrade complementary mRNA, reducing protein expression

What are the properties of siRNA and miRNA?

• siRNAs are exogenous, double-stranded, and fully complementary to mRNA (causing degradation)

• miRNAs are endogenous, single-stranded, processed from primary transcripts, and may block translation with partial complementarity.

What are Dicer and RISC?

• Dicer is an RNase III enzyme that cleaves dsRNA into short siRNAs or miRNAs

• RISC (RNA-induced silencing complex) unwinds these and uses one strand to target complementary mRNAs.

What are the basic steps in RNA interference?

1. dsRNA recognized by Dicer

2. Dicer cuts it into siRNAs

3. siRNAs associate with RISC

4. RISC unwinds the siRNA and binds to complementary mRNA

5. mRNA is degraded or translation is inhibited.

What features of bacterial plasmids are useful in the expression of eukaryotic genes?

Plasmids are small, have their own origin of replication, can be present in high copy numbers, carry selectable markers, and allow easy insertion of foreign DNA

What are restriction enzymes and how are they used in generating recombinant DNA?

Restriction enzymes are endonucleases that cut DNA at specific sequences

• generating sticky ends that can be used to insert foreign DNA into plasmids

What is cDNA and why is it used in bacterial expression systems?

cDNA is a complementary DNA version of mature mRNA (exons only)

• It is used in bacterial expression systems because it lacks introns, which bacteria cannot splice

How is the appropriate double-stranded cDNA generated for a gene of interest?

mRNA is reverse transcribed into single-stranded cDNA using reverse transcriptase, then a second strand is synthesized using DNA polymerase

What features need to be added to eukaryotic genes for successful expression in bacteria?

• A bacterial promoter for transcription

• Shine-Dalgarno sequence for translation, and the absence of introns (use of cDNA) are needed

What elements are needed in each reaction tube during Sanger sequencing?

• Template DNA

• labeled primer

• DNA polymerase

• four dNTPs

• one of the dideoxy analogs (ddNTPs) are needed to terminate the polymerization

How can controlled termination be used to identify DNA sequences?

Controlled termination using ddNTPs results in fragments of different lengths

• which can be separated by gel electrophoresis or detected using fluorescent tags

What is the basic idea of Next-Generation Sequencing (NGS) and pyrosequencing?

• NGS involves parallel processing of DNA fragments to generate massive amounts of sequencing data.

• Pyrosequencing detects light produced during nucleotide incorporation.

What are the components of a CRISPR-Cas system?

A CRISPR-Cas system consists of a single guide RNA (sgRNA) for targeting DNA and a Cas protein (usually Cas9) that cuts the target DNA.

What happens to the DNA targeted by CRISPR-Cas?

The targeted DNA is cleaved by Cas9, creating a double-strand break

What determines whether target DNA is excised or replaced in CRISPR-Cas?

If no repair template is provided, the DNA is excised. If a template for homology-directed repair is provided, the DNA can be replaced