Chapter 12 - Manipulating DNA: Tools and Techniques Flashcards
gene technology
a broad field which includes analysis of DNA as wells genetic engineering and other forms of genetic modification
genetic engineering
artificial manipulation of genes
adding or subtracting genes, or changing the way genes work
genetically modified organisms (GMO)
benefits of gene technology
increasing crop and livestock production preventing and fighting disease reducing pollution and waste producing new products detecting and preventing crime
action of restriction enzymes
cut DNA fragments at precise locations
action of electrophoresis
seperate fragments by size
action of probes
find particular DNA fragments
action of ligase enzyme
join DNA fragments
action of vectors
transport DNA into cells
action of gene cloning
obtain multiple copies of gene
restriction enzymes (endonuclease)
used to cut up DNA molecules at very precise recognition sites
‘molecular scalpels’
recognition site
specific site for the restriction enzyme that can cut DNA at that point
cutting site
sticky ends
restriction enzymes that cut leaving an overhang
two different fragments cut by the same restriction enzyme have identical sticky ends and are able to join by what?
base paring
annealing
blunt ends
restriction enzymes that cut leaving no overhang
able to join with any other blunt end fragment
ligation
pieces of DNA are joined together using the enzyme DNA ligase
DNA ligase
enzyme involved in the joining of DNA fragments
recombinant DNA
DNA that has recombined from different sources by DNA ligase
steps of ligation
2 pieces of DNA are cut using the same restriction enzyme
the 2 different DNA fragments are attracted to each other by weak hydrogen bonds
other end of the foreign DNA is attracted to the remaining sticky end of plasmid
annealing
when 2 matching sticky ends com together and are joined by base pairing
forms linear or circular (plasmid)
plasmid
circular fragment of DNA
present in many copies in bacteria cells
gene cloning
process of making large quantities of desired piece of DNA once it has been isolated
vector
self-replication DNA molecule used to transit a gene from one organism to another
steps of gene cloning
gene of interest isolated from human
appropriate plasmid vector is isolated from bacteria
gene and plasmid treated with same restriction enzyme to produce identical sticky ends
cuts plasmid in one place, cuts DNA in many places (only one has specific gene)
DNA ligase used to bond sticky ends of plasmid and DNA, forming a recombinant DNA plasmid
introduced into bacterial cell and cell division occurs
DNA amplification / PCR (polymerase chain reaction)
creates vast quantities of DNA identical to trace samples
artificial DNA replication
DNA polymerase
enzyme that controls the process of DNA replication
steps of PCR
target DNA is obtained
DNA is denatured to single stranded by heating for 5mins at 98
primers (mRNA) annealed to each DNA strand
sample cooled to 60
DNA polymerase binds to primers on each side of DNA
this enzyme synthesises a complementary strand of DNA using free nucleotides
after one cycle: 2 copies of original sample
primers
short, single-stranded DNA or RNA sequences complementary to a part of a specific DNA fragment
gel electrophoresis
used to seperate large molecules on basis of size and electric charge
steps of gel electrophoresis
prepare: restriction digest: DNA cut into smaller pieces
DNA placed into wells and covered with buffer solution
molecules move towards electrode depending on charge
DNA negatively charged (phosphates)
smaller fragments go further down
DNA profiling (fingerprinting)
technique for genetic analysis which identifies the variation found in the DNA of individuals
gel electrophoresis
DNA probe
a small fragment of nucleic acid that is labeled with an enzyme, radioactive tag or fluorescent dye tag
work by binding to a specific sequence on DNA
DNA probes may be used for
presence of specific alleles
approximate location of gene on chromosome
genetic fingerprint of a person (paternity testing)
Transformation
using genetic engineering techniques, concerned with the movement of genes from one species to another
transgenic organism
organism that develops from a cell into which foreign DNA has been introduced
recombinant DNA technology
allows direct modification of an organisms genome
allows traits to be introduced
transformation is used for:
crops
production of herbicide resistant plants
enhancement of livestock
treatment of human defects
DNA sequencing
used to determine the nucleotide (base) sequence of DNA
gene therapy
the application of gene technology to correct or replace defective genes
target cancer, treat genetic disorders, provide relief for infectious diseases
not inherited when on somatic cells
