chap 3- DNA manipulation techniques and applications Flashcards
what are restriction endonucleases
enzymes that cut DNA at a specific recognition site
what are restriction enzymes used for
to cut DNA into smaller more usable fragments and isolate particular regions of interest (single genes)
When do restriction enzymes occur naturally
in bacteria
what do restriction enzymes form
bacterial cells defense system, targeting foreign DNA that may enter the cell
What are sticky-ends
when restriction enzymes leave DNA fragments with overhanging ends
what are blunt ends
when restriction enzymes leave clean-cut ends
how do sticky end restriction enzymes cut the DNA backbone
at a different location on each strand within the recognition site
how do blunt end restriction enzymes cut the DNA backbone
by cutting the sugar-phosphate backbone on both strands of the DNA molecule at the same location within the recognition site
What are ligases
a group of enzymes that join fragments of DNA or RNA and create a phosphodiester bond between them.
What are polymerases
enzymes that catalyse the formation of polymers, in particular nucleic acids
why does DNA polymerase act
to produce identical copies of DNA
what does RNA polymerase do
acts to assemble RNA. In cells, RNA polymerase synthesizes mRNA, rRNA and tRNA by transcription of genes.
what does PCR stand for
Polymerase Chain Reaction
what is PCR used for
to obtain larger amounts of DNA when there is only a small amount
In PCR: what does the DNA sample provide
a target sequence of DNA that is to be occupied and amplified.
In PCR what do primers provide
a starting point for DNA to be built on
what are primers
short nucleic acid sequences
In PCR what are free nucleotides used for
added to solution in order for DNA to be built
In PCR what does taq polymerase do
binds to primer and begins building DNA from the free nucleotides
In PCR what is the buffer solution for
provides suitable chemical environment for activity of polymerase
what is the significance of Taq polymerase for PCR
its extracted from bacteria that lives in hot springs meaning it can function in high temps without degrading
What are the 3 steps of PCR
denaturation. annealing. extension
what happens during denaturation
sample heated to 95 degrees. breaks hydrogen bonds between the strands of DNA to obtain single strands of DNA.
what happens during annealing
temp is reduced to 50-60 degrees. allows primers to bind to complementary base sequences through hydrogen bonds
what happens during extension
temp increased to 72 degrees. allows taq polymerase to attach to the primers on the DNA strands. the taq polymerase moves along each strand adding free nucleotides to form double stranded DNA.
in each PCR cycle how much is the number of double stranded copies of the DNA increased by
double the amount
what does gel electrophoresis do
separates fragments of negatively charged DNA by length
what can gel electrophoresis technique be used for
DNA screening, fingerprinting for forensics, paternity cases, confirming correct gene has been amplified by PCR
what does DNA profiling do
identifies one individual from another individual
what is DNA profiling used for
identifying perpetrators of a crime, identifier of bodies and to settle paternity disputes
what are non-coding DNA sections
regions that are not transcribed or translated into a protein
what does STR stand for
short tandem repeats
what does DNA profiling rely on
an individuals unique DNA, specifically the non-coding sections of DNA which vary widely between individuals
what are STRS
sections of 2-6 bases located within the non-coding sections of DNA, located on specific chromosomes can be used to identify a person uniquely from someone else
what is the process of DNA profiling
DNA sample extracted using restriction enzymes
STR’s amplified using PCR with specific primers for each STR
Gel electrophoresis differentiates STR’s
DNA sample is a match if lengths of particular STR’s are all the same size
what does CRISPR stand for
Clustered regularly interspaced short palindromic repeats
What does regularly interspaced mean
spaces occur at regular intervals
what does palindromic mean
short sequence of letters that reads the same forwards and backwards
What is spacer DNA
sections of DNA from foreign substances such as viruses, bacteriophages or plasmids that have been incorporated into the bacterium’s genome.
what are Cas genes
genes that encode Cas proteins which are helicases that cause DNA to unwind enabling endonucleases to cut the DNA
what is adaptive immunity
immunity an organism acquires after exposure to a pathogen
what is a bacteriophage
a type of virus that infects its host bacterial cell by injecting its genetic material into the host cell. takes over host cell until it kills the cell
what is the role of Cas1 proteins
break apart bacteriophage DNA and store a section of that DNA in the CRISPR locus
What is the bacterial CRISPR immune system
DNa is transcribed to make tracrRNA and pre-crRNA along with ranscribing and translating Cas proteins
what does CRISPR array consist of
duplicate sequences of palindromic repeats belonging to the bacterial genome flanked by spacers belonging to foreign genetic material
what is pre-CRISPR RNA (pre-crRNA)
RNA with repeat palindromes and spacers that is transcribed in one strand
what is tracer RNA (tracrRNA)
has sections that are complimentary to the palindrome repeats enabling it to anneal to the pre-crRNA strand within the Cas9 protein
how does Cas9 complex work to defend bacteria from viral DNA
nuclease enzyme will search for a short specific sequence unique to viral genome (PAM). then the viral DNA will unwind. one target DNA is located endonuclease will snip both strands of DNA just a few bases upstream from the PAM
what does PAM stand for
protospacer adjacent motifs
where is PAM found
after the DNA sequence that the Cas9 nuclease wants to cut
What do PAMs do
help bacteria distinguish their own DNA from viral DNA
what is guide RNA (sgRNA)
synthetic version of the DNA strand being fed through Cas9
what are some CRISPR applications
effective with helping sickle cell anaemia, cystic fibrosis, muscular dystrophy
What are plasmids
small circular pieces of double stranded extrachromosomal DNA molecules that are physically separated from chromosomal DNA and can replicate independently of chromosomal DNA.
where are plasmids found
bacteria
what is a recombinant plasmid
a plasmid that has been altered in the laboratory through the insertion of a foreign gene
what is the origin of replication
a DNA sequence that allows initiation of replication within a plasmid through the use of host cells replication machinery
What is an antibiotic resistance gene
allows for selection of plasmid containing bacteria
what is a selectable marker
enables selection of transformed plasmids by differentiating them from non-transformed plasmids
what is a promotor
DNA sequence upstream from gene to be expressed that RNA polymerase attaches to and begins protein synthesis of that gene
what are restriction sites
specific DNA sequences that act as cut sites for restriction enzymes
what is the inserted gene
gene that has been inserted into plasmid
what is transformation
process used by bacterial cells to introduce foreign material into the cell, this foreign material can form into a plasmid
what is a vector
any vehicle that is used to ferry a desired DNA sequence into a host cell as part of a molecular cloning procedure
what is complementary DNA (cDNA)
a DNA copy of a mature mRNA molecule produced by reverse transcriptase using a reverse transcriptase enzyme
how is cDNA made
from mature mRNA cause its had its introns removed and therefore only contains exons
What are the two ways you can transform bacterial cells
heat shock and electroporation
what is electroporation
where a pulse of electricity is used to briefly open pores in the cell membrane of bacteria so plasmids can be taken up into the bacteria
what is heat shock treatment
bacterial cells and plasmids placed in ice-cold solution containing calcium ions. temp is rapidly increased creating pressure difference between out/inside of cell inducing the formation of pores in the plasma membrane and allowing the supercoiled plasmid to enter. bacteria is then allowed recovery time
how can recombinant plasmids be identified
bacteria cultures are placed onto agar plates with an antibiotic that recombinant plasmids are resistant to. bacterial cells that have been transformed through the uptake of the recombinant plasmid will survive, the ones that haven’t will die
What is a GMO
genetically modified organism
what is a transgenic organism
an organism that’s genome has been altered through the insertion of a gene from another type of organism
what is insulin
a peptide hormone that promotes the uptake of sugar from the bloodstream and its storage in muscle or adipose tissue.
what produces insulin
B-cells of islets of Langerhans of the pancreas
what does insulin consist of
two polypeptide chains, an A chain consisting of 21 amino acids and a B chain consisting of 30 amino acids
what happens during option 1: placement of insulin gene within beta-galactosidase (B-gal)
when b-gal is functioning, bacterial colonies that are plated on a medium of x-gal will be blue in colour. when insulin is placed within the B-gal gene, the B-gal isn’t produced and therefore cant break down X-gal and bacterial colonies that have taken up the recombinant plasmid containing the insulin gene will be white in colour allowing for easy identification
what happens in option 2: placement of insulin gene next to B-gal
promotes transcription of the insulin gene, which then allows for the production of functional insulin.
this is because when lactose is added to the E.Coli cell it promotes the synthesis of B-gal and cause the insulin gene is next to the B-gal gene, insulin gets synthesized as well
