Genetics Lecture 20 Material

What is biotechnology?

The use of molecular techniques to develop new products

What are molecular techniques used to do?

Isolate, recombine, and amplify genes

What is recombinant DNA technology?

A set of molecular techniques for locating, altering, recombining, and studying specific pieces of DNA

What are restriction enzymes?

Site-specific endonucleases that recognize and cut DNA at specific nucleotide sequences

What are engineered nucleases?

Nucleases designed to cleave DNA at predetermined sequences

What is CRISPR-Cas genome editing?

A facile method for editing genomes across the phylogenetic spectrum

What is the most useful class of restriction enzymes?

Type II

What are restriction enzymes apart of?

Bacterium's restriction modification system

What does a bacterium's restriction modification system do?

Defend against viruses

What is each restriction enzyme paired with?

Restriction methylase

What does restriction methylase do?

Methylates the recognition sequence on DNA

What does methylation of a recognition system do?

Blocks the cleavage by the restriction enzyme to protect the bacterium's own DNA from being digested

What does the first three letters of a restriction enzyme represent?

The bacterial species from which the enzyme was isolated

What would a fourth letter in a restriction enzyme name mean?

Strain of bacteria from which the enzyme was isolated

What do the roman numerals in a restriction enzyme name mean?

Identification of different enzymes from the same species

What are the three types of ends produced from restriction cleavage?

Cohesive, overhangs, and blunt

What is a cohesive end?

Fragments with short, single-stranded overhanging endsWh

What are the two types of overhangs?

3' or 5'

What are blunt ends?

Even-length ends from both single strands

What are two names for cohesive ends?

Sticky or overhanging

What are two examples of engineered nucleases?

Zinc-finger and transcription activator-like effector

What does CRISPR stand for?

Clustered Regularly Interspersed Short Palindromic Repeats

What is CRISPR derived from?

Bacteriophage and plasmid genomes

What does a CRISPR array consist of?

Palindromes and spacers

What two components combine to provide defense against invasion by specific foreign DNA molecules

CRISPR RNAs (cRNAs) and Cas (CRISPR-associated)

How does the effector complex of CRISPR-Cas work?

Binds to foreign DNA through base pairing and CAs cleaves the DNA, making it dysfunctional

CRISPR-Cas9 is a technique used for precisely editing the _____

Genome

What is the advantage of the long sequence recognized by sgRNA?

Edits can be almost anywhere in the genomeW

What are the two main advantages of CRISPR-Cas9 editing?

Easier and less expensive

How has CRISPR-Cas9 been modified?

Introduce single-stranded cuts and sticky ends, which improves efficiency of inserting DNA fragments

What are the disadvantages of CRISPR-Cas9 editing?

Off-target cleavage, genetic mosaics, and difficulty introducing

What are the concerns with CRISPR-Cas9 editing?

Genetically modified humans raise ethical questions, safety due to off-target cuts, potential danger of editing animals/plants and releasing into wildW

How does gel electrophoresis separate DNA molecules?

Based on size

What charge does DNA possess?

Negative

What charge is DNA attracted to?

Positive

Shorter DNA fragments travel _____ up the gel?

Farther

Longer DNA fragments travel _____ up the gel?

Shorter

What can southern blotting detect?

A single restriction fragment in a complex mixture of restriction fragments

What are the three types of gel blots?

Southern, Northern, and Western

What is a Southern blot?

DNA on a gel that is transferred to a filter, and then hybridized with a DNA or RNA probe

What is a Northern blot?

RNA on a gel that is transferred to a filter, and then hybridized with a DNA or RNA probe

What is a Western blot?

Protein on a gel is transferred to a filter, and then an antibody is used to detect a specific protein

What does PCR stand for?

Polymerase Chain Reaction

What does PCR do?

Amplify specific DNA fragments

What are the limitations of PCR?

Sequence of target DNA must be known, amplification of contaminants, accuracy, and length of PCR products

What are the applications of PCR?

Quantitatively determine the amount of DNA amplified as the reaction proceed

What are the 3 steps in PCR?

Denature, Anneal, and Extension

What does the denaturation step of PCR do?

Separates two strands of DNA

What does the annealing step of PCR do?

Short single-stranded primers binds to complementary sequences

What does the extension step of PCR do?

DNA polymerase synthesizes new DNA strands

After the three steps of PCR, what is the product of PCR?

Two new, double-stranded DNA molecules

Each time the cycle of PCR repeated, the amount of target DNA ____

Doubles

What is molecular cloning?

A specific piece of DNA is amplified using a host cell

What are used to replicate the desired piece of DNA in the host cell?

Cloning vectors

What are plasmids?

Circular DNA molecules from bacteria

What are linkers?

Synthetic DNA fragments containing restriction sites

How is foreign DNA inserted into plasmids?

Restriction enzymes and DNA ligase

How are cells screened for recombinant plasmids?

Selectable markers are used to confirm whether or not the cells have been transformed

What are the qualities of an ideal vector?

Origin of replication, one or more selectable markers, and one or more unique restriction sites

Why must a cloning vector have an origin of replication?

So that it is replicated along with the DNA that it carries

What is an example of a typical cloning vector?

pUC19

What are 5 examples of other types of cloning vectors?

Phages, cosmids, bacterical artificial chromosomes (BACs), Yeast Artificial Chromosomes, Ti plasmids

How does one ensure transcription and translation occurred?

Inserting a foreign gene into an expression vector

What do expression vectors contain?

Operon sequences that allow inserted DNA to be transcribed and translated. As well as sequences that regulate the desired gene

What plasmid can be used to transfer genes into plants?

Ti plasmid

Where is the Ti plasmid from?

Agrobacterium tumefaciens

How does the Ti plasmid integrate into a plant through natural gene transfer?

The agrobacterium invades the plant at a wound, part of the Ti plasmid is transferred to the plant cell, where it integrates into one of the plant chromosomes

How is Ti plasmid integrated into plants through genetic engineering?

Foreign DNA is inserted into a plasmid vector and transferred to Agrobacterium tumefaciens with the Ti plaasmid, which is then transferred to a plant cell

What is bacillus thuringiensis?

A gram-positive bacterium that produces several substances toxic to insects

What are the most notable insecticidal chemicals in bacillus thuringiensis?

Crystalline proteins called delta-endotoxins

The bacillus thuringiensis gene was isolated from bacteria and transferred to what type of plants?

Tobacco

What is a DNA library?

A collection of clones containing all the DNA fragments from one source

What is in situ hybridization?

DNA hybridization probes can be used to locate chromosomal or cellular location of a gene or its RNA product

What is cDNA?

A DNA copy of mRNA

What is cDNA synthesized?

mRNA is purified and then a single-stranded copy of the mRNA template is made using reverse transcriptase. The RNA is eliminated and the remaining single stranded cDNA is made double stranded using DNA polymerase, which is then cloned.