Topic 14 Flashcards
restriction enzymes
- also called endonuclease- tool uses to cut DNA
- have ability to cut DNA molecules at very precise sequences - recognition sequences
- once recognised- enzyme binds to DNA and cuts at precise and predictable way
- restriction enzymes occur naturally in bacteria, thought to have evolved as defense mechanisms against viruses
- named after bacterial species isolated from
- some cut DNA at directly opposite points- produce blunt ends
- others cut one strand at one point, and another at non-directly opposite point- overhanging ends are sticky- complimentary
Crispr-Cas9
- complex comprising Cas9 endonuclease and single guid RNA
- cuts at precise sequence and used to edit genes
- endonuclease complex occurring naturally in bacteria- used to chop DNA of invading viruses
- two components required: sgRNA- locates and binds to target sequence of DNA, Cas9 endonuclease- unwinds and cuts DNA
- PAM sequence- lies downstream of target region of non-target DNA strand- recognition of PAM by Cas9 destabilised DNA- allows sgRNA to be inserted- CAs9 doesn’t function without PAM sequence
gene knock in (gene editing)
- new DNA sequence is inserted into genome
- allows faulty gene to be corrected with normal correct sequence
gene knock out (gene silencing)
- cell’s normal repair process attempts to mend DNA
- errors occur resulting in insertion or deletion of nucleotide
- frame shift mutation causes nucleotide sequence reading, either disabling gene sequence or producing STOP signal
electrophoresis
-DNA is negatively charged-phosphate groups in sugar phosphate backbone
-sorts DNA according to size
standards- DNA fragments of known length-used to compare size of sample DNA
-DNA moves from negative terminal to positive terminal- negatively charged
-shortest DNA move most quickly through gel- found further from starting point
-DNA fragments made visible through fluorescent dye/radioactive probe
-end result is parallel bands different distances down gel
probes
single strand of DNA/RNA with base sequence complimentary to one of strands of target DNA
- labelled with fluorescent tag/radioactive so that location of probe and target DNA can be seen
- will bind to target DNA sequence by CBP, identifying presence/location of target DNA sequence
- typically used for position of gene on chromosome
- presence of allele of gene
how probe is used in southern blotting
DNA of interest extracted from living/dead organism
- cut using restriction enzymes
- fragments separated using gel electrophoresis
- DNA transferred by blotting to filter/membrane
- probes added to filter/membrane- if complimentary sequence is found, probes hybridise with other strand
ligation
- process of joining DNA fragments cut using restriction enzymes
- DNA ligase, catalyses joining pieces of double stranded DNA
- can produce longer linear sequence/one circular molecule of DNA
- two pieces of DNA of different origins joined using restriction enzymes/DNA ligase- recombinant
- bacterial plasmids used in recombinant technology
- plasmids replicate independently of bacterias chromosomes
- carry antibiotic resistant markers
ligation process
two pieces DNA cut using same restriction enzymes, produce sticky ends
- when two sticky ends come together, they anneal- stabilising molecule to allow permanent join
- DNA fragments are permanently joined by DNA ligase-catalyses phosphodiester bond between sugar and phosphate of backbone, producing recombinant DNA
Making DNA fragments
synthesise DNA from nucleotide building blocks
-uses DNA synthesiser
-base sequence of DNA must be known
-joins nucleotides in pre-defined order
-doesn’t require template strand or DNA polymerase- products used as probes/primers
make copy of DNA using mRNA template
-double stranded DNA from eukaryotic organism isolated
-DNA is transcribed- producing primary RNA molecules
-introns removed by restriction enzymes to form mature mRNA- codes for proteins
-mRNA is extracted and purified
-reverse transcriptase is added to make single stranded DNA molecule complimentary to mRNA
-second strand made using first as template, adding enzyme DNA polymerase
why remove introns
- makes DNA shorter and easier to insert into plasmids
- large amounts of non-coding not made by PCR
- allows bacteria to properly translate human gene- due to bacterial DNA not containing introns
DNA amplification-PCR
-creates large quantities of trace amounts of DNA
-cheap, quick and accurate and uses thermal cycler
-also uses target DNA, free DNA nucleotides, primers, Taq polymerase
Steps
Denaturation- heat to around 90 degrees- weakens hydrogen bonds and DNA disassociates into single strands
Annealing- mixture is cooled to around 50 degrees- primers are added and form hydrogen bonds with regions at either end of target DNA
Extension- mixture heated to 72 degrees- Taq polymerase uses primers as starting point and extends so that two complete DNA strands are formed.
plasmids
self-replicating DNA molecule
-acts as vectors
properties of all vectors
- be able to replicate independently inside host organism
- have one or more sites at which restriction enzymes can cut
- have some kind of genetic marker that allows easy identification
bacterial transformation process
- gene of interest isolated from cells
- both DNA and plasmid are cut with same restriction enzyme- produce identical sticky ends
- restriction enzyme cuts plasmid DNA at single recognition sequence- disrupts tetracycline resistance gene
- DNA fragments are mixed together- attracted by CBP
- DNA ligase is added to bond sticky ends
- recombinant plasmid is introduced into bacterial culture
- under right conditions, some bacteria take up plasmid by transformation
