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Flashcards in Chapter 11 Deck (65)

What is genetic engineering

It is using "in vitro" techniques to manipulate and alter genetic material


What was genetic enginerring first developed with

Bacteria and phage


What are some basic tools of molecular/DNA/gene cloning

Restriction enzymes, PCR, Gel electrophoresis, nucleic acid hybridization and probes, molecular cloning and cloning vectors


What are restriction enzymes

They recognize specific DNA sequences and cut DNA at those sites. Widespread in prokaryotes but rare in eukaryotes. Are used to protect prokaryotes from hostile foreign DNA (viral genomes) and is important for in vitro DNA manipulation


What are the three classes of restriction enzymes

Type I and III: Don't cleave at their recognition site and are not very useful for cloning
Type II: Cleave DNA within their recognition sequence and are the most useful for specific DNA manipulation and cloning


What are the recognition sequences that restriction enzymes recognize

They recognize inverted repeat sequences (usually homodimers, palindromes)


What do restriction enzymes produce after they cleave the sequence

They produce sticky or blunt ends of a DNA fragment. Each restriction enzyme always produces the same ends everywhere it cuts


How do restriction enzymes destroy foreign DNA

By cutting it into pieces


What are sticky ends

When the restriction enzyme cuts the dsDNA to have staggered ends with overhangs


What are modification enzymes

They protect cell's DNA from its own restriction enzymes. They chemically modify nucleotides in restriction recognition sequence. Modification is often methylation of DNA


What is Gel electrophoresis

It is used to separate DNA molecules base on size. It uses an electrical field to separate charged molecules in a gel matrix (DNA is negatively charged)


What are the gels made out of in gel electrophoresis

Gels are made of agarose (polysaccharide isolated from seaweed) or polyarylamide (a chemical polymer)


Describe the process of gel electrophoresis

Nucleic acids migrate through the gel toward the positive electrode due to their negatively charged phosphate groups and molecular sieving slows the larger molecules


What is ethidium bromide used for

DNA in gels can be stained with ethidium bromide or other dyes and visualized under UV light


How are restriction enzymes used with gel electrophoresis

The location of restriction sites is determined by DNA sequence. Each restriction enzyme will cut the DNA at the same site so you will get the same fragments. If given a random piece of DNA and the fragments match up with known fragments, you can identify the DNA.. DNA cut with different restriction enzymes will produce different size fragments


What is a restriction map

A map of the location of restriction enzyme cut sites on a segment of DNA (used to identify DNA molecules)


What are restriction fragments

DNA fragments resulting from digestion with restriction enzymes, these can be cloned into a vector by ligation


What is nucleic acid hybridization

Base pairing of single strands of DNA or RNA from two different sources with the same or very similar sequences


What is a nucleic acid probe

Segment of known single-stranded DNA or RNA that is used in hybridization. Can be synthetic DNA or a cloned restriction fragment. The probe is "labeled" so it can be detected


How is the nucleic acid probe labeled

The "label" can be a radioactive atom, specific chemical group, fluorescent molecule etc


What is a southern blot

A hybridization procedure where DNA fragments separated by size in a gel is transferred to special membrane, and then hybridized with a labeled RNA or DNA probe. It is used to find restriction fragments with specific genes


What is a northern blot

RNA is in a gel instead of DNA


What will the probe hybridize with in the southern blot

The probe hybridizes with only homologous fragments on the blot to which it can base pair


What is molecular cloning

Isolation and incorporation of a piece of DNA into a vector so it can be replicated and manipulated. Originally developed in bacteria.


Why are bacteria useful in molecular cloning

Bacteria are a major cloning host, easy, fast, less expensive than other types of host cells


What are the three main steps of gene cloning

1. Isolation of source DNA fragment
2. Insertion of DNA fragment into cloning vector
3. Introduction of cloned DNA into host organism


List possible sources of source DNA

1. Total genomic DNA
2. cDNA
3. PCR-amplified DNA
4. Synthetic DNA fragment


How is source DNA isolated

Source DNA is often digested with restriction enzymes to produce stick ends for ligation into a vector


What is genomic DNA

Total genomic DNA (to make a library of clones)


What is cDNA

It is copied DNA from mRNA. Total mRNA for a library and specific gene determined by primer used to make the cDNA


What is PCR-amplified DNA

DNA fragments containing a specific gene


Describe the inerstion of DNA fragment into cloning vector

The vector must replicate in the host cells. Most vectors are derived from plasmids or viruses and the DNA is generally inserted in vitro


What is DNA ligase

An enzyme that joins two DNA molecules. It is required for DNA replication and repair and can work with sticky or blunt ends. Requires ATP to work


What is overlap PCR

PCR technique for joining DNA molecules and allows for more precise constructions


Describe the introduction of cloned DNA into host organism

Transformation is often used to get recombinant DNA into host cells, but conjugation and transduction also can be used


What are possible recombinant DNA sources

Specific cloned fragment, gene library or shotgun cloning


What is a gene library

It is a mixture of clones, each containing a different randomly cloned DNA fragment from a total genome or other source


What is shotgun cloning

Gene libraries made by cloning random genome fragments


What are possible outcomes after the cloning procedure

Only some transformed host cells will contain desired cloned gene, while other cells will have nothing, just the vector, or the wrong cloned DNA.


What do you have to do to find your cloned gene

1. Initial screen for host cells that contain cloning vector. Antibiotic resistance for plasmid vectors, plaque formation for viral vectors
2. Find clones with your gene of interest. If working with a gene library composed of thousands to millions of cloned DNA fragments, then you must have a specific probe or assay for the gene or gene product that you want.


What are proper probes or assays when wanting to find your clones with your gene of interest

Hybridization with DNA or RNA probes, antibody binding to protein product or assay for protein activity


Describe the major steps for gene cloning

First you start with foreign DNA, then it is cut with a restriction enzyme and results in sticky ends. Then you add it into a vector that has also been cut with the same restriction enzyme. Then you add DNA ligase to form recombinant molecules which is then introduced into a host


Describe the major steps for finding the right clone

First you will have transformant colonies growing on your plate.. Then the plate will be replicated with a membrane filter and then you will either:
1. (Protein product) partially lyse cells, add specific antibody, add agent to detect bound antibody in radiolabeled form
2. (DNA) Lyse bacteria and denature DNA, add RNA or DNA probe (radioactive) and wash out unbound radioactivity
Then detect radioactivity for positive clones


What is mutagensis

Making changes to genes to produce products with different acitivites


Describe synthetic DNA

Oligonucleotides of up to 100 bases can be made easily. Multiple oligonucleotides can be ligated together or assembled with PCT to make larger fragments and whole genes/gene libraries can be constructed.


What is synthesized DNA used for

Synthesized DNA is used for PCR primers (15-30 bases), labeled probes, or for site-directed mutagnesis


What are conventional mutagens

They produce mutations at random


What is site-directed mutagensis

They are performed in vitro and introduces mutations at a precise location into cloned genes. Used to alter specific amino acids in a protein to change function, gives insight into protein structure and function


Describe the major steps of site-directed mutagensis

First part of a source material is cloned into a single stranded DNA. Then you add syntehtic oligonucleotide with one base mismatch and the single strand will be extended with DNA polymerase resulting in a mutant cell (transformation). The mutant cell can be cloned and selected


What is cassette mutagensis

A DNA segment can be replaced by a synthetic DNA fragment called cassette or cartridge. DNA cassettes often contain an antibiotic-resistance gene for selection


What is gene disruption in knockout mutations

It results when cassettes are inserted into the middle of the gene. Knockout mutants are produced by recombination between mutated cloned gene and its homologous gene on the chromosome


Describe the major steps of gene disruption by cassette mutagnesis

First a gene X on a plasmid is cut and a kanamycin cassette is placed in the middle of the gene before its ligated back together. Then the plasmid is cut with a different restriction enzyme and transformed into a cell with wild-type gene X. The recombination and selection for kanamycin-resistant cells will take place leaving you with a gene X knockout, knockout mutants


What are reporter genes

They encode proteins that are easy to detect and assay


What are some examples of reporter genes

lacZ: encodes beta-galatosidase
luxAB: encodes luciferase enzyme (produces light)
gfp: encodes GFP


What are gene fusions

A promoter or gene of interest can be fused with a reporter gene to monitor gene regulation under different conditions


Describe the construction of gene fusions

First you have a two different strands of DNA:
1. Target gene with a promoter and coding sequence
2. Reporter gene with a promoter and coding sequence
Then you cut and ligate them together resulting in a Gene fusion with a promoter from the target gene and the coding sequence of the reporter gene. Used to monitor the gene regulation of the target gene by using the target gene promoter with an easily detectable reporter gene encoding sequence


Describe plasmids as cloning vectors

They are small and easy to isolate DNA. Have an origin of replication, contain multiple copy number: multiple copies of cloned gene per cell makes more cloned DNA/protein product. Have selectable markers (antibiotic resistance) and can be transferred into host cells by transformation or electroporation


On plasmids what is the polylinker

The polylinker is the "multiple cloning site" that is within the lacZ gene. It contains restriction enzyme cut sutes and the cloning sites are restriction sites present only once in a vector


Describe the process of using plasmids as cloning vectors with lacZ gene

The insertional INACTIVATION of lacZ gene indicates presence of cloned DNA fragment. Inactivated lacZ cannot produce beta-galatosidaze enzyme, so colonies are white. Activated lacZ can produce beta galatosidase enzyme that will cleave colorless X-gal and produce a blue dye. Can determine your positive vectors with blue/white screening. Blue colonies don't have foreign DNA and white colonies do have foreign DNA cloned into plasmid


Describe ideal host cells

Capable of rapid growth in inexpensive medium, nonpathogenic, capable of carrying or incorporating foreign DNA, genetically stable in culture, allow replication of cloning vectors and capable of expressing cloned genes. Bacteria


What are shuttle vectors

Vectors that are stably maintained in two or more unrelated host organisms: E.coli and yeast or E.coli and human cells. Bacterial plasmids can be engineered to function in eukaryotes by adding a eukaryotic origin of replication and centromere sequence or the ability to recombine into a host chromosome


What are expression vectors

They allow controlled expression of cloned genes. They allow for high levels of protein expression, contain strong promoters, and transcription terminators that are used to prevent expression of other genes


How is mRNA from cloned gene efficiently translated into host cells

For expression in bacteria you need a bacterial ribosome binding site (Shine-Dalgarno), correct codon usage with tRNA, and eukaryotic genes must have introns removed for expression in bacteria, usually by cloning cDNA copied from mature mRNA. This is because bacteria do not perform splicing


Describe bacteriophage lambda as a cloning vector

A modified lambda phage can make a good cloning vector because it has well-understood biology, can hold larger amounts of DNA than most plasmids, and DNA can be efficientyly packged into phage particles in vitro


What are bacterial artificial chromosomes (BACs)

They are constructed from the F plasmid and can carry alrge fragments of DNA. Often used as a vector for genomic cloning and sequencing