Genetics 244 - Recombinant DNA technology

(Chapter 20)

What determines how frequently a restriction enzyme will cut?

The number of base pairs in the recognition site and its specific sequence.

Type I RE

Will cut far from recognition site/sequence

Type II RE

Will cut at the recognition site

Palindrome

A sequence of DNA that reads the same forwards and backwards, typically found in the recognition sites of restriction enzymes.

Cohesive (sticky) ends

Can base pair with complementary single stranded ends on other DNA fragments cut using the same RE, and anneal to complementary sequence by H-bonds

Which enzymes cleave sticky ends?

EcoRI and HindIII

Which enzymes cleave blunt ends?

EcoRV and Smal

What key properties do vectors have to have? (4)

They must contain various unique RE sites, must be capable of replicating in host cells independent of host cells chromosomes, must have a selectable marker gene or a reporter gene and should be easy to recover from host cell

Types of vectors: (6)

Plasmids, phages, cosmids, shuttles, YACs and BACs

What are plasmids?

Small, extrachromosomal DNA molecules within a cell that are physically separated from chromosomal DNA and can replicate independently

Multiple cloning site/polylinker

A short segment of DNA which contains many restriction sites for commonly used restriction enzymes

Selectable marker gene

Genes that provide resistance to antibiotics (E.g: ampR for ampicillin resistance)

Blue colonies:

Functional lacZ → non-recombinant plasmid

White colonies:

Non-functional lacZ → recombinant plasmid

Cloning vector vs expression vector

A cloning vector is a small DNA molecule that carries a foreign DNA fragment into the host cell, whereas an expression vector is a vector that facilitates the expression of genes and production of proteins.

Genomic libraries

The entire genome of an organism in many fragments

What is the objective of complementary DNA (cDNA) libraries?

To provide a “snapshot” or catalogue of just the genes that were transcriptionally active at a given time.

Polymerase Chain Reaction (PCR)

A rapid method of obtaining multiple and specific copies of a particular region of DNA

What does the PCR require? (5)

Target dsDNA, DNA polymerase, primers (forward and reverse), dNTPs and Mg2+ in buffer solution

What can the restriction enzyme mapping (RE Mapping) report?

Number of, order of and distances between restriction-enzyme cleavage sites along a cloned segment of DNA

in situ Hybridisation involves:

the hybridisation of a probe directly into a chromosome or RNA without blotting

What is DNA sequencing?

The process of determining the order of the 4 nucleotide bases in a DNA molecule

What is the most commonly used method for DNA sequencing?

Sanger Sequencing

What is the difference between PCR and Sanger?

PCR: generates dsDNA, uses two primers (F + R), and dNTPs

Sanger: generates ssDNA, only one of the DNA strands is copied, uses only one primer and dNTPs and ddNTPs

How does PacBio work?

It works by attaching ss molecules of DNA to be sequenced to a single molecule of DNA Poly anchored to a substrate, this is then visualised in REAL-TIME!

Why would Nanpore sequencing be chosen over Illuminia?

Longer contigs, the assembly process is more accurate, and does not require any amplification.

What is a contig?

A contig is a set of DNA segments or sequences that overlap in a way that provides a contiguous representation of a genomic region.

What is the point of gene targeting?

To manipulate a specific allele, locus or base sequence to learn about the functions of a gene of interest

A knockout (KO)

An example of a loss-of-function mutation

Conditional knockouts

Allows one to control the particular time in an animal’s development that a target gene is disrupted

What system is used to make conditional knockouts?

The Cre-lox system

What is the difference between transgenic and knockout animals?

Transgenic animals have a foreign gene inserted into their genome, while knockout animals have a gene inactivated or removed 

What does CRISPR-CAS stand for?

Clustered Regulatory Interspaced Short Palindromic Repeats - CRISPR Associated protein