Topic 11: Biotechnology

DNA technologies

techniques used to isolate, purify, analyze, and manipulate DNA sequences

What do scientists use DNA technologies for?

both basic and applied research

genetic engineering

the use of DNA technologies to alter genes for practical purposes, part of biotechnology

biotechnology

any technique applied to biological systems or living organisms to make or modify products or processes for a specific purpose

what does biotechnology include?

• genomics

• bioinformatics tools

genomics

the characterization of the whole genome

bioinformatics tools

the application of math and computer science to biological data

gene cloning

when DNA cloning involves a gene

What is a common method of gene cloning?

• inserting the gene of interest into a plasmid which produces recombinant DNA molecules 

• the plasmids are then inserted into bacteria, which replicate the recombinant DNA as they grow and divide

genetically alter organisms

any organism that has its genome altered to change a genetic trait or traits

genetically modified organisms (GMOs)

organisms that have their genomes specifically engineered to introduce or change a genetically controlled trait

What do GMOs contain?

recombinant DNA

recombinant DNA 

DNA fragments from 2 or more different sources that have been joined together to form a single molecule

basic research

studies gene structure or function, including how its expression is regulated, and the nature of the gene’s product 

applied research 

cloned DNA used for medical, forensic, agricultural, or commercial applications 

restriction endonucleases (restriction enzymes)

• bacterial enzymes used to join 2 DNA molecules from different sources

• recognize specific DNA sequences (restriction sites) and cut the DNA at specific locations within those sites 

restriction fragments

the DNA fragments produced by a restriction enzyme

sticky ends

single-stranded ends of DNA fragments that H bond with complementary sticky ends on other DNA molecules cut with the same restriction enzyme 

ligation 

DNA ligase seals the sugar-phosphate backbones of the DNA strands 

what are bacterial plasmids examples of?

cloning vectors 

cloning vectors

DNA molecules in which a DNA fragment is inserted to form a recombinant DNA molecule for the purpose of cloning

which 2 genes are plasmid cloning vectors engineered with to locate bacteria?

• amp^R gene

• lacZ⁺ gene

amp^R gene

encodes an enzyme that breaks down the antibiotic ampicillin 

lacZ⁺ gene

encodes B-galactosidase, which hydrolyzes the sugar lactose, acts as a restriction site, DNA fragments and plasmids are both cut within this gene with the same restriction enzyme 

What are DNA molecules transformed into?

ampicillin-sensitive, lacZ⁺ E. coli 

What happens after DNA molecules are transformed into ampicillin-sensitive, lacZ⁺ E. coli?

they are spread on a plate containing ampicillin and the B-galactosidase synthetic substrate x-gal

How are bacteria that have been transformed with recombinant plasmids identified?

with blue-white screening

polymerase chain reaction (PCR)

produces an extremely large number of copies of a specific DNA sequence without having to clone the sequence in a host organism (replicates DNA replication) 

What are the primers used in PCR designed to do?

designed to isolate the sequence of interest by cycling 20-30 times through a series of steps, amplifying the target sequence, producing millions of copies

What enzyme does PCR exclude?

helicase, which is replaced by a raise in temperature

gel electrophoresis 

a technique that separates DNA, RNA, and protein molecules in a gel subjected to an electric field, DNA has a negative charge and moves towards the positive end

What is gel electrophoresis based on?

based on size (mass) (smaller fragments move easier through the gel and thus move farther) , electrical charge, or other properties 

How can PCR results be compared?

by using agarose gel electrophoresis, compares the movement of segments to a standard DNA marker ladder, sees how far the segments have traveled compared to the standard used 

agarose gel electrophoresis

the size of the amplified DNA is determined by comparing the position of the DNA band with the positions of bands of the DNA ladder 

transgenic

organisms that receive genes from an external source (transgenes)

What are there some ethical concerns with genetic engineering?

fear that these methods may produce toxic/damaging foods or release dangerous and uncontrollable organisms 

how is E. coli engineered to make a foreign protein?

• the protein-coding sequence of a gene is inserted into an expression vector (plasmid) which contains regulatory sequences that allow transcription and translation of a gene

• recombinant plasmid becomes E. coli

• the inserted gene is expressed in E. coli, transcribed, and translated to make the encoded eukaryotic protein

• the protein is extracted from bacterial cells and purified, or purified frm the culture medium 

What do most eukaryotic protein-coding genes have that bacterial and most archaeal protein-coding genes don’t have"?

introns

After mRNA has been processed in eukaryotes, a double-stranded DNA copy can be made by….

reverse transcription, a complementary DNA (cDNA) is made by the enzyme reverse transcriptase and used in reverse transcriptase-PCR

gene targeting

the knocking out, replacement, or addition of a gene in a genome

In PCR testing, what happens at 96 C?

denaturing - the double strand becomes a single strand

In PCR testing, what happens at 55-65 C?

annealing - primers match to the complementary sequences at either end

In PCR testing, what happens at 72 C?

extension - the target sequence is replicated making 2 copies of the original strand

totipotent

can turn into fully functioning organisms (our very first cell)

stem cells

capable of undergoing many divisions in an undifferentiated state, and also have the ability to differentiate into specialized cells 

adult stem cells 

function to replace specialized cells in various tissues and organs (multipotent) 

embryonic stem cells

(pluripotent) found in an early-stage embryo (blastocyst) and can differentiate into all of the tissue types of the embryo

knockout mouse

a homozygous recessive that receives 2 copies of a gene altered to a nonfunctional state (transgene)

How do you make a knockout mouse?

• introducing a nonfunctional gene (transgene) to the embryonic stem cells

• eventually germ cells contain the transgene

What do CRISPR and cas genes do together?

encode for an immune system against foreign bacteriophages and plasmids in bacterial and archaeal cells

programmable RNA-guided genome editing system

the natural CRISPR-case system modification that is used for research purposes

gene therapy

the introduction of a normal gene into particular cell lines to correct genetic disorders

germline gene therapy

the experimental introduction of a gene into germline cells of an animal, not allowed in humans

somatic gene therapy

somatic cells are cultured and transformed with an expression vector containing a transgene, modified genes are reintroduced int into the body

restriction fragment length polymorphism (RFLP)

restriction enzyme-generated DNA fragments of different lengths from the same region of the genome, typically analyzed using agarose gel electrophoresis

single-nucleotide polymorphism 

a single base pair mutation (ex. sickle cell anemia), it has two alleles and the frequency of the rarer gene has to be less than 1% 

What can RFLP help determine?

to identify markers to see if someone has inherited a certain disease

DNA fingerprinting (profiling) 

a technique used to distinguish between individuals of the same species using DNA samples, commonly used in forensics and parternity testing 

What is used to analyze DNA fingerprinting?

pcr analysis and gel electrophoresis

short tandem repeats

short sequence of DNA
repeated in a sequence (2-6 BPs long), the
number of repeats at each loci varies between individuals in a population