Foundations of Biology Exam 5: Chapter 20

recombinant DNA technology

A variety of techniques for isolating specific DNA fragments and linking them to different regions of DNA or adding them to a differential host organism; allows researchers to recombine specific DNA sequences from any organism to create DNA not found in nature

DNA cloning

any of several techniques for producing many identical copies of a particular gene or other DNA sequence

plasmid

small circular DNA molecules most common in prokaryotes; often modified with recombinant DNA as a cloning vector

cloning vector

a modified plasmid or virus used to transfer recombinant genes into cells

restriction endonulease (enzyme)

any class of bacterial enzymes that cut DNA at a specific base-pair sequence (recognition site)

DNA ligase

seals added recombinant DNA segments to the old (original) DNA strands

biotechnology

the engineering of genes, cells, and organisms for basic research and practical purposes; recombinant DNA is a cornerstone of this

genetically modified organism (GMO)

a plant or animal that has had specific changes introduced into its DNA using genetic engineering methods

gene knockout

a loss-of-function mutation that most often has been engineered rather than naturally occurring; classic method to deduce function by observing what happens when the gene doesn't work; DNA recombination allows researchers to be specific

function; expressed

genetically modified animals are made when researchers are trying to determine gene ________, or to determine when genes are ________ (2 different answers)

transgenic

a plant or animal whose genome contains DNA introduced from another individual, often from a different species

agrobacterium tumefacins

bacteria that infects many different plant and does so by transferring part of the plasmid it carries (Ti plasmid) into the host plant; when making a transgenic plant tumor inducing genes for the T-DNA is removed and replaced with the genes researchers want to introduce

GMO plant formation from a single cell

1. foreign gene is placed on a nutrient-rich medium and allowed to grow into a mass of cells called a callus

2. callus transferred to another medium that promoted development into a tiny plant with roots and shoots that can be grown in soil

3. new plant is propagated and its seeds are used for breeding

gene therapy

the treatment of an inherited disease either by reintroducing a normal form of the gene or correcting an improperly functioning gene; usually easier in principle than in practice

CAR-T therapy

modified immune system cells that recognize tumor cells; engineered outside the body and infused; mostly effective for blood cancers

requirements for gene therapy success

1. the disease must be due to defects in a single gene

2. the sequence of the wild-type allele must be known

3. there must be a way to introduce it into affected individuals and have it expressed in the correct tissues, at the correct time, and in the correct amount

4. if a dominant allele, there must be a way to replace the defective allele with one that functions normally, not simply adding a functional allele

modified viruses

_____ ______ are the most common cloning vector to deliver therapeutic genes; their genomes are altered to include the gene and to disable replication of this in the target cells

ex vivo

one of the primary approaches to gene therapy; "outside the body"; cells that require the therapeutic gene are removed from the patient and then infected with the viral gene therapy vector; vectors for this method typically integrate the gene into the patients genome

in vivo

one of the primary approaches to gene therapy; "in the body"; the viral gene delivery vector is injected into the bloodstream and the virus is transported throughout the patients body through the bloodstream

somatic gene therapy

gene therapy that genetically modifies body cells, not germ cells, so the genetic modification cannot be inherited in future generations

heritable cell gene therapy

gene therapy that results in genetic modification of germ cells, with the consequence that the genetic modification can be inherited in the future generations; until recently this was forbidden; no technology precise enough; should only be considered for diseases caused by defects in a single gene and only when genome modification is a last resort

CRISPR-Cas

a way to edit genomes based on an RNA (sgRNA) associated with a Cas protein that cuts DNA at sites complementary to the sgRNA; discovered in prokaryotic cells and modified to become an indispensable part of biology's molecular toolkit

CRISPR

acronym for a locus in bacteria and archaea that is compromised of repeat sequences separated by spacer sequences derived from viruses that have infected the cells or its ancestors; produces crRNAs that with a Cas protein, are the basis for current genome editing methods

original steps of CRISPR-Cas stepup

1. the CRISPR locus is transcribed into long pre-crRNA

2. pre-crRNA is processed into a set of shorter RNA fragments (crRNA) that each contains a spacer region that is a RNA copy of part of a viral genome flanked by short stretches of bacterially encoded repeat sequences

3. a crRNA binds to the complementary DNA sequence of an invading virus, but cannot act alone

4. non-coding tracrRNA pair with the end of crRNA

5. new crRNA-tracrRNA molecule binds to a Cas proteins

Cas protein

an endonuclease that cuts DNA at site complimentary to an RNA bound to it; are a core element to the CRISPR-Cas system

sgRNA

single guided RNA; a fusion of crRNA and tracrRNA designed to simplify the CRISPR-Cas system for practical use

common method of CRISPR-Cas

1. mix sgRNA with plasmid DNA that contains the gene for Cas9

2. introduce the mixture into a cell

3. once Cas9 is expressed, the protein associated with sgRNA which guides Cas9 to a complimentary target sequence in the genome

4. Cas9 makes double-stranded DNA cuts in the DNA

5. either NHEJ or HDR occur

nonhomologus end joining (NHEJ)

does not require homology; method of genome editing that joins two broken strands of DNA back together, although always with insertions or deletions; if cut is made in a coding sequence- reading frame will likely shift; if cut is made in regulatory sequence- the sequence will be altered

homology directed repair (HDR)

method of genome editing that if an intact segment of DNA is available that spans the region of the double strand break, the segment is exchanged for the broken DNA; trick is to introduce the new DNA fragments with the CRISPR-Cas machinery

polymerase chain reaction (PCR)

a technique for rapidly generating many identical copies of a specific stretch of DNA; works by multiple rounds of DNA replication, starting from primers designed to amplify particular DNA sequences; much faster than plasmid cloning

primers

to amplify a particular DNA sequence, a researcher needs some information about the surrounding DNA to design ______

primers

short lengths of single-stranded DNA that matches sequences on either side of the region to be amplified; allow DNA synthesis reaction at the core of PCR to begin

heating; high

PCR depends on separating complementary strands of DNA at each amplification cycle which is done by _______ the DNA solution; because normal DNA polymerase cannot function at these ______ temps DNA polymerase from heat resistant microbes are used

DNA fingerprinting

any technique for identifying individuals based on the unique features of their genome

short tandem repeats (STRs)

consist of simple repeating units from two to about eight nucleotides long; occur outside of protein coding regions; core of DNA fingerprinting; the number of these in a genome varies widely among individuals

quantitative reverse transcriptase-PCR (qRT-PCR)

a form of PCR in which an RNA is first converted to a cDNA using reverse transcriptase, then PCR is used to amplify the cDNA in cycles that are continuously monitored for the amount of amplified cDNA; technique allows for measuring the amount of starting RNA in a sample

environmental DNA (eDNA)

DNA shed into the environment and analyzed, typically using PCR, to learn about the composition of population organisms resident in that environment

the value of learning a gene or genome nucleotide sequence

1. the genetic code can be used to infer the amino acid sequence of the gene's protein product; amino acid sequence often provides clues to protein function

2. comparing sequences often reveals why alleles vary in function

3. evolutionary relationships can be inferred by comparing genome sequences in different species or between individuals of the same species

dideoxy sequencing

a technique for determining the nucleotide sequence of DNA; relies on the use of dideoxynucleoside triphosphates (ddNRTs) which terminate DNA replication; in its original form, many identical copies of a DNA strand were required

next-generation sequencing (NGS)

general term used to describe DNA sequencing technologies that are faster and cheaper than dideoxy sequencing; millions of different DNAs can be amplified and sequenced in a single run

shotgun sequencing

a method of sequencing genomes that is based on breaking the genome into small pieces, sequencing each piece, and then figuring out how the pieces are connected; fragments are of varying size

genomic DNA

DNA that is derived directly from cells, in contrast to cDNA; fragments of this DNA are expected to overlap in many regions after shotgun sequencing

de novo genome assembly

using a computer algorithm, a process of genome assembly that pieces together fragments of DNA produced by next-generation sequencing

reference genome

a genome sequence established for a species that serves as a point of comparison for genome sequences from other individuals of the same species; 1st genome of a species sequenced

bioinformatics

a discipline at the intersection of biology, computer science, and statistics concerned with the storage, analysis, and presentation of biological data, particularly with nucleotide and amino acid sequences in the field of genomics

genomics

the field of study concerned with sequencing, interpreting, and comparing whole genomes from different organisms

genome annotation

the process of analyzing a genome sequence to identify key features such as genes, regulatory sequences, and splice sites; easier to do in prokaryotes; uses bioinformatics

twenty

when doing genome annotation and looking for protein coding sequences, the computer system will predict the RNA sequences of the two DNA strands and the three different reading frames per RNA sequence; random sequences should contain a stop codon coded for every ______ codons

open reading frame

a sequence of DNA that, if converted into a RNA sequence and translated as a set of non-overlapping codons, would produce a polypeptide of substantial size; discovery of one provides evidence for the existence of a protein-coding gene; finding these in eukaryotes is harder because coding sequences are divided into intron and exons

homology

similarities among organisms of different species due to shared ancestry; features that exhibit such similarity (ex. DNA sequences, proteins, body parts) are said to be this; variations are from mutations that are allowed to thrive

expressed sequence tags (ESTs)

a cDNA from a portion of transcribed gene that is used to find the gene's physical location in the genome and to confirm that a region of DNA is transcribed; the cDNA is the tag

genome-wide association study (GWAS)

a method of location genes by finding associations between a particular phenotype and a polymorphic genetic marker (almost always SNP) in populations; purpose is to look for association between phenotypes of interest and DNA sequences at known locations in the genome; in humans usually disease vs. non-disease phenotypes

genetic markers

a genetic locus that can be identified and traced in populations by molecular techniques and by distinctive visible phenotype; DNA must come in at least two common sequence variants to work as this

single nucleotide polymorphism (SNP)

a site on a chromosome where different versions of the chromosome have different nucleotides; can be used as a genetic marker to track the inheritance of nearby genes; a site in DNA that varies at a single base pair; if this has a know location and is inherited with a particular trait, we can infer the gene must lie near the know location

principles of prokaryotic genomes

1. compact: introns are rare, most coding sequences are uninterrupted, little space between genes, use operons, have few regulatory sequences

2. bacterial species that live in a wide variety of habitats and use a wide array of nutrients for food have large genomes; parasitic species that steal from host have small genomes

3. most genes in one species are not shared widely with others

4. genome size and content vary extensively with species

5. genomes frequently rearrange during evolution

6. a significant amount of genome has been acquired by lateral gene transfer

prokaryotic genome

genome size increases, number of genes increases

lateral gene transfer

transfer of DNA between two different species

two criteria to identify laterally transferred DNA

1. the gene of interest if more than similar to genes in distantly related species than in closely related species

2. the proportion of G-C base pairs to A-T base pairs in a particular gene or series of genes is markedly different from the base composition of the rest of the genome

transformation

when bacteria and archaea take up raw pieces of DNA that are transferred from the environment

metagenomics

the inventory of all the genes in a community or ecosystem created by sequencing, analyzing, and comparing the genomes of the components organisms; often refers to the study of microbial communities

repeated DNA sequences

_______ ____ _________ partially explain the immense variation in eukaryotic genome size

transposible elements

any of several kinds of DNA sequences that are capable of moving themselves, or copies of themselves, to other locations in the genome; similar to retrovirus; passed from parents to offspring, but do not travel outside the host

long interspersed nuclear elements (LINE)

any of the most abundant class of transposable elements in human genomes, can create copies of itself and insert then elsewhere in the genome; can cause mutations that can be negative, neutral, or positive

gene duplication

the formation of an additional copy of a gene, often by misalignment of chromosomes during crossing over; an important evolutionary process in creating new genes

gene famliy

a set of genes whose DNA sequences are similar because they arose by duplication of an ancestral gene followed by divergence of the sequence due to accumulation of random mutations

unequal crossing over

an error in crossing over during meiosis I in which the two non-sister chromatids match up at different sites; results in gene duplication in one chromatid and gene loss in the other

duplication and divergence

an evolutionary process in which new genes are creates by duplication of an original gene followed by divergence of the copied gene sequences allow one ore both copies to acquire a new function

pseudogenes

a DNA sequence that closely resembles a functional gene but no longer functions; arises by duplication of the functional gene followed by inactivation due to mutation

long noncoding RNA (lncRNA)

an abundant class of transcribed RNA molecules that are > 200 nucleotides in length; do not code for a protein, and in some cases function in the regulation of gene expression

functional genomics

the study of how, when, and where specific genes are expressed within the genome and how their products interact to produce a functional organism; focus in genome-wide patterns of gene expression

transcriptomics

the complete set of gene transcripts in a particular cell; the most common way to learn genome-wide patterns of expression

DNA microarrays

an early method of transcriptomics; a set of single-stranded DNA fragments, representing thousands of different genes that are attached to a surface such as a glass slide; can be used to determine which genes are expressed in different cell types, under different conditions, or at different developmental times

deep sequencing (RNA-seg)

a method to learn the types of mRNAs or DNA sequences present in cells and their relative amounts; involves preparing a sequencing cDNAs prepared form the cell or tissue; most common method for transcriptomics

spatial-omics

looks at the expression of genes in single cells at know locations within a tissue; compares gene expression cell-to-cell of cells in the same tissue

proteomics

the systemic study of the full set of proteins (the proteome) in a cell or tissue, including their localization, functions, regulation, and other functions

proteome

the complete set of proteins produced by a particular cell type