DNA Manipulation Flashcards
DNA manipulation
using DNA in a lab for our benefit
Gene editing
Process of making targeted changes to a specific section of an organism’s genome, including adding, deleting or altering specific DNA sequence
Restriction Enzymes (endonucleases)
Enzymes that cut DNA at a specific location (restriction site)
Palindrom
a sequence of DNA, that you can read from 5’ to 3’ and vice versa
Polymerase
joins base pairs together
Helicase
Unzips DNA
Gyrase
Unwinds DNA
RNA polymerase
makes a polymer of RNA
DNA polymerase
makes a polymer of DNA
Reverse transcriptase
makes cDNA from mRNA
endonuclease
cuts DNA at a specific recognition sequence
Ligase
Joins DNA segments with “sticky ends”
Gene probes
Single-stranded DNA or RNA complementary base sequences to target DNA. Attached with fluorescent markers. DNA must denature and then anneal
CRISPR
cas9 are enzymes in the cell that can cut up DNA that matches a little piece of DNA that they hold inside of themselves.
Cas9 will take the RNA transcribed from the DNA and hold the RNA in the enzyme to travel around the cell to find DNA that is complementary to the RNA.
This can be used to cut DNA at any place we want which lets us alter the genome
Ligase
Opposite of endonucleases. Enzyme that can stick two single strands of DNA together to form one longer strand
DNA amplification
Using the technique called polymerase chain reaction (PCR), researchers can create vast quantities of DNA identical to trace samples
Polymerase Chain Reaction
Technique by which one can copy segments of a DNA sample repeatedly to form multiple copies of the same DNA for further analysis
PCR process
- mixture of DNA fragments heated to seperate strands (92 degrees)
- temperature cool to allow primer to bind to DNA
- temperature increased again to allow Taq DNA polymerase to start building complementary strand
- process is repeated over and over
Gel Electrophoresis
method of separating fragments of DNA, usually according to size
Gel Electrophoresis process
- sheet of gel used to seperate fragments
- DNA fragments added to one end of gel
- electric current run through gel
- electric current run through gel
- DNA is negatively charged
Plasmid
double-stranded circular DNA molecules separate from the bacterial chromosome. Replicate independently. Usually has its own marker gene
Recombinant plasmid
plasmids that we put foreign genes into. Some plasmids may not incorporate the gene of interest because they may reseal before the gene of interest can bind. This is because the complementary sticky ends are attracted to themselves
Transformed bacteria
bacteria that have incorporated the recombinant plasmid
Electroporation
using electrical currents to open pores and make the membrane permeable
heat shock
freezing for 1 min, heating for 1 min to open pores, more permeable to accept plasmid
Vectors
a self-replicating DNA molecule used to transmit a gene from one organism to another.
Must have the following properties:
- Be able to replicate inside their host organism
- Have one or more sites at which a restriction enzyme can cut
- Have genetic marker that allows them to be easily identified
DNA Sequencing
To identify the sequence of DNA
DNA Sequencing process
- DNA strand is heated, hydrogen bonds between bases break
- DNA is allowed to cool allowing primer to bind
- DNA polymerase builds complementary strands
- repeat for all four nucleotides
DNA profiling
To identify DNA from different individuals
DNA profiling process
small tandem repeats: Sites in the DNA where many copies of a short DNA sequence are joined end to end
- when cut with the same endonuclease, DNA fragments will be different lengths
- gel electrophoresis shows different lengths of DNA fragments due to STR
Gene cloning
Making multiple copies of a gene using prokaryotes.
Gene cloning process
- endonuclease that leave sticky are used to cut before and after a gene
- gene fragment is inserted into another species for cloning
Genetic Screening
Refers to an organised program of testing of groups or a population to achieve early detection or exclusion of inherited disorders. Testing someone for the presence of an allele known to cause disease.
Positive results of genetic screening
- Can adjust lifestyle to prevent the condition
- Early treatment prevention
- Planning for life
Negative results of genetic screening
- False positive results
- Discrimination for jobs, insurance
- Costs, resources used in testing
