Chapter 21: Genetic Technologies and Genomics

CRISPR/Cas9

a bacterial system that can be used either to produce a mutation in a specific gene or to correct a mutation that is already present

CRISPR/CAS Mice

Silence a gene that encodes tyrosinase, enzyme needed for pigment production

Recombinant DNA technology

Use of laboratory techniques to bring together fragments of DNA from multiple sources

Gene Cloning

Process of making multiple copies of a particular gene.

-Make large amounts of specific genes DNA (to study DNA directly or use DNA as a tool).

-Make large amount of gene product (study structure/function of a protein, other uses such as medicine).

Genomics

Molecular analysis of the entire genome of a species

Vector DNA

Carrier for the DNA segment to be cloned.

-May carry small segment of chromosomal DNA

-It can replicate, making copies, when introduced into living cell

Plasmids

Vector. Small circular pieces of DNA found naturally in many strains of bacteria

Viral Vectors

Derived from viruses which infect living cells and propagate themselves using the host cell's machinery

Restriction Enzymes

Cut DNA.

-Made naturally by bacteria as a protection against bacteriophages.

-Cut at specific known restriction sites in DNA

-May produce sticky ends.

Recombinant Vector

Vector containing a piece of chromosomal DNA

Restriction sites

A specific sequence on a DNA strand that is recognized and cut by a restriction enzyme.

Selectable Marker

A gene, such as one encoding resistance to an antibiotic, that can be used to identify (select) cells that contain recombinant DNA from among a large population of untransformed cells.

-ampR

ampR

ampicillin resistance gene.

-Only cells with the plasmid grow on plates treated with ampicillin

lacZ System

To eliminate recircularized empty vectors.

-Cleaves colorless X-Gal into blue dye.

-Reciruclaized plasmids form blue colonies

-bacteria containing desired recombinant vectors form white colonies

DNA Library

Collection of many recombinant vectors each with a fragment of chromosomal DNA. A collection of DNA fragments that have been cloned into vectors so that researchers can identify and isolate the DNA fragments that interest them for further study

Genomic Library

a type of DNA library in which the inserts are derived from chromosomal DNA

cDNA Library

a type of DNA library which uses reverse transcriptase to make complementary DNA (cDNA) from mRNA

Reverse Transcriptase

An enzyme encoded by some certain viruses (retroviruses) that generates complementary DNA (cDNA) from an RNA template

Electrophoresis

Technique used to separate macromolecules on a gel

-Used to separate DNA/proteins.

-Often used to evaluate the results of a cloning experiment.

-Separate molecules based on charge, size/length, and mass.

polymerase chain reaction (PCR)

Make many copies of DNA in a defined region without vectors or host cells. Used for forensics.

dideoxy chain termination method

a DNA sequencing method in which target DNA is denatured and annealed to an oligonucleotide primer, which is then extended by DNA polymerase using a mixture of deoxynucleotide triphosphates (normal dNTPs) and chain-terminating dideoxynucleotide triphosphates (ddNTPs)

ddNTPs

Missing 3'-OH and cause chain to terminate

DNA Microarry

glass slides that have single-stranded DNA

-Used to show expression of genes

-High fluorescence intensity in spot indicates high levels of cDNA in sample

DNA Microarry Application

Cell-specific gene expression, gene regulation, elucidation of metabolic pathways, tumor profiling, genetic variation (disease causing alleles), microbial strain identification

CRISPER-Cas Process

sgRNA binds to Cas9, guides it to the target gene, Cas9 makes a double stranded break

Two different DNA repair events

• End joining may lead to a small deletion

• If the researcher adds donor DNA homologous to the break region a specific mutation can be put into the gene

tracrRNA

Trans Activating Crispr RNA, required for binding to Cas9 protein

crRNA

RNA derived from spacer element that matches target DNA to cut

sgRNA

An RNA molecule engineered to be complementary to the target to be edited (tracrRNA + crRNA)

Bacterial/Archaeal Genomes

Studying these genomes important because: bacteria cause disease, can apply knowledge to more complex organisms, origin of first eukaryotic cell probably involved union or archeal and bacterial cell, bacteria often used as tools in research

Venter and Smith

Completely sequenced a bacterial genome: haemophilus influenzae

Shotgun DNA Sequencing

Break DNA into thousands of small fragments

Use computer to assemble fragments based on their overlapping parts

Sequencing Eukaryotic Genome

benefit from identifying and characterizing genes in model organisms

- more information to identify and treat human disease

- improved strains of agricultural species

-way to establish evolutionary relationships

Gene Duplication

Provides raw material for the addition of new genes into a species' genome.

-create homologous genes

Homologous Genes

Two or more genes that are derived from the same ancestral gene.

-Over many generations each version of the gene accumulates different mutations

Paralogs

homologous genes within a single species

Gene Family

Two or more paralogous genes that carry out related functions

Pseudogenes

Genes that have been produced by gene duplication but have accumulated mutations that make them nonfunctional.

-Not transcribed into RNA

Human Genome Project

Identify all human genes, sequence entire human genome, develop technology, analyze genomes of model organisms, develop legal, ethical, and social programs addressing the results

Repetitive Sequences

Segments of DNA repeated multiple times in a genome

Transposable Elements

Genetic element that has the ability to move (transpose) from one site on a chromosome to another.

-type of repetitive sequence "jumping genes"

Moderately Repetitive Sequence

Few hundred to several thousand times

- rRNA genes

-multiple origins of replication

-role in gene transcription and translation

Highly Repetitive Sequences

Tens of thousands or millions of times

-some clustered together in a tandem array

-others interspersed throughout genome

-Alu sequences in humans/primates

Alu Sequence

Family of mobile genetic elements that comprises about 10% of the human genome; this short, repetitive sequence is no longer mobile on its own, but requires enzymes encoded by other elements to transpose.

Noncoding/Coding DNA

98% genome noncoding, 2% genome coding (exons of structural genes, genes for rRNA and tRNA)

Transposition

when a short segment of DNA moves from original site to a new site

Barbara McClintock

discovered transposable elements

DNA Transposons

"jumping genes" remove themselves from one place in the genome and move to a new site

-Both ends have inverted repeats

Inverted Repeats

DNA sequences that are identical but run in opposite directions

Transposase

An enzyme that catalyzes the insertion of DNA segments into other DNA molecules

-Binds to the end of a transposon and catalyzes its movement to another part of the genome by a cut and paste mechanism or a replicative transposition mechanism

Cut-and-Paste Mechanism

-Transposase recognizes inverted repeat and then removes sequence from original site

-Complex moves to new location where transposase inserts it into the chromosome

Retrotransposons

A transposable element that moves within a genome by means of an RNA intermediate

-Common only in eukaryotes

Integrase

the enzyme responsible for integrating viral DNA into the host cell's DNA (inserts retrotransposon into DNA)

cDNA

complementary DNA, DNA copy of mRNA

taq polymerase

A heat-stable form of DNA polymerase extracted from bacteria that live in hot environments, such as hot springs, that is used during PCR technique