Restriction enzymes and DNA cutting Flashcards
how is polyploidy genetic variation induced
need to add a chemical
what is genetic engineering
manipulation of DNA of organisms for the purposes of, changing inheritance or the in vitro manufacture of gene products
what is a mutation
any change in genetic information relative to reference ‘wild type’ genome
what is a mutagen
any agent causing mutations
how do mutations with mutagens occur
randomly
what occurs in genetic engineering
enable us to take genes out
genetic material analysed
DNA can be altered
DNA can be re-inserted in organism
how can DNA be cut
shaking DNA violently, but this will cause random breaking
DNA can be cut using enzymes in more specific and targeted way
what are the two enzyme types
Endonucleases
Exonucleases
what is an enzyme
a biological catalyst; all biochemical reactions are controlled by enzymes and in general, each enzyme controls each separate reaction
what is a restriction enzyme
alternative name for endonuclease
what is an exonuclease
cleaves nucleotides one at a time from the end (exo) of an polynucleotide chain
what did Werner Arber discover
restriction enzymes
what did Daniel Nathans discover
Pioneered the application of restriction for the construction of genetic maps
what did Hamilton Smith discover
Showed that restriction enzyme cuts DNA in the middle of a specific sequence
what happened in 1978
Arber, Nathans and Smith won a Nobel Prize
how was HindlI discovered
is the first restriction enzyme discovered accidentally in 1970; while studying how the bacterium Haemophilus influenzae defends itself to the virus
what can the bacteria Haemophilus influenzae do
break down the DNA from the phage and any other foreign DNA; yet its own DNA is not damaged
what does base Y mean
C+T
what does base R mean
G+A
what can exonucleases be used for
- Removal of oligonucleotides before a PCR reaction
- Removal of chromsomal DNA in plasmid preparations
- Removal of DNA in RNA preparations
- Generation of single strand DNA (ssDNA) from linear double strand DNA (dsDNA)
what is recombinant DNA technology
creation of a new combination of DNA segments that are not found together naturally
what are the applications of recombinant DNA technology
Basic research on control of gene expression
Forensic medicine
Biotechnology - generate new products
how do restriction endonucleases work
Cleave phosphodiester bonds of nucleic acids at an internal site
highly specific; can recognise 4 bases pair (HaeIII), 6bp(EcoRI), 8bp (NotI) or more
restriction sites that are recognised are usually palindromic
what is the use of a restriction endonuclease
Cut DNA internally and in a more specific and targeted way; their role is vital for the use of Genetic Engineering
what is a palindrome
a sequence of units that can be read the same way in either direction
what is the frequency of enzyme recognition
4n
n = number of bases in recognition sequence
if each position of the recognition site has 4 possible bases what is the probability of finding any one base
1/4
where does Haelll recognise a segment
segment of 4 bases
5’ GC/CC 3’
3’ CC/GG 5’
where does Smal recognise a segment
segment of 6 bases
5’ CCC/GGG 3’
3’ GGG/CCC 5’
where does Notl recognise a segment
segment of 8 bases
5’ GC/GGCCGC 3’
3’ CGCCGG/CG 5’
what are blunt ends
end of a DNA molecule where both stands ends in a base pair
what is an overhang
stretch of unpaired nucleotides in the end of a DNA molecule, they can be in either strand
what are sticky ends
overhangs of more than one nucleotide at the end of a DNA molecule; also called cohesive ends
how are blunt ends generated
shearing DNA or by cutting with blunt end restriction endonucleases (eg. HindII or SmaI)
what does deoxynucleotide terminal transferase do
add short poly-T sequences at the 3’-ends of an opened vector.
Add short poly-A sequences to the 3’-ends of DNA to be cloned
what is used to seal the blunt ends
ligase
how is a sticky end formed
DNA strands not cut at the centre of the restriction site
staggered cutting leaves overhanging segment of 4 base pairs
5’ G| AA|TTC 3’
3’ CTTAA| G 5’
what do sticky ends form
transient double helical structures
what is used to join sticky ends
ligase
what happens if different DNA molecules are mixed
some ligation will generate new combinations
which was the first enzyme discovered
Hindll
which enzyme can create sticky or blunt ends
endonucleases
what does a restriction map show
positions of restriction sites in sequence of DNA
