CHAPTER 2: DNA MANIPULATION Flashcards
1
Q
DNA polymerase
in DNA manipulation
A
uses DNA polymerase to accurately copy a DNA template
2
Q
endonucleases - restriction enzymes
A
- restriction enzymes like endonucleases cut DNA at specific recognition sequences known as restriction sites, splitting DNA into smaller fragments
- restriction sites is a particular order of nucleotides
endonucleases make one incision on each of the 2 complementary strands of DNA
3
Q
sticky ends
A
- endonucleases cut one strand at one point but cut the second strand at a point that is not directly opposite → causes an overhang to form
- DNA ligase enzyme connects the single-stranded DNA together via the sugar-phosphate back bones
4
Q
which pairs faster, sticky or blunt
A
- pieces of DNA with sticky ends pair faster than pieces of DNA with blunt ends
- sticky ends allow for complementary base pairing so the pieces are held together by weak hydrogen bonds and can be acted on by the DNA ligase enzyme.
5
Q
blunt ends
A
- endonuclease cuts the 2 strands of DNA molecule at points DIRECTLY OPPOSITE each other to produce cut end
- DNA fragments are joined directly together through the use of DNA ligase
6
Q
DNA ligase
A
an enzyme known as ligases catalyses the joining of pieces of double-stranded DNA at their sugar-phosphate bones
7
Q
gel electrophoresis
A
- a method used to separate DNA fragments based on size (length, measured in bp)
- the phosphate group of nucleotides is negatively charged
- negatively charged DNA moved towards the positive terminal (b/c it is negatively charged)
- DNA is loaded into a gel that acts like a sieve to allow SMALLER DNA fragments through more quickly than larger ones
- the result of gel electrophoresis is a series of PARALLEL BANDS of DNA fragments at differing distances down the gel each band can contain millions of DNA molecules of the same size
8
Q
applications of gel electrophoresis
A
- people have DIFFERENT DNA SEQUENCES, so when endonucleases are used, the DNA will be cut at DIFFERENT LOCATIONS and DNA fragments of different lengths will result
- gel electrophoresis can identify people in:
- forensic investigations
- mass disasters
- paternity testing
- identifying animals
9
Q
mitochondrial DNA
A
- not all DNA is used in gel electrophoresis
* nuclear or mitochondrial DNA can be used - compared to nuclear DNA, mtDNA
- is inherited via the MATERNAL LINE
- does not recombine during reproduction → LESS VARIABLE
- is present in LARGER AMOUNTS
10
Q
dna profiling - short tandem repeats
dna profiling - short tandem repeats
A
- chromosomal sites (non coding region) where many copies of a short DNA sequence are joined end-to-end; the number of repeats is variable between unrelated people
- STRs are 2-5 bp in length and repeated over and over
- the number of REPEATS VARIES between people and each variation is a distinct allele
- we have 2 copies of each STR - one from mother and one from father
11
Q
types of DNA used for DNA profiles
A
- to develop DNA fingerprints or DNA profiles, regions with high variation between individuals are used.
- Short Tandem Repeats (STRs) from nuclear DNA
STRs can identify individuals - Hypervariable regions (HVRs) in mtDNA
mtDNA can identify relationships/when fewer cells are available
- Short Tandem Repeats (STRs) from nuclear DNA
12
Q
what are plasmids
A
- circular, double stranded DNA that can reproduce independently
- can be taken up by bacterial cells
- many are used as vectors to transport foreign DNA into bacterial cells to transform them
- they have multiple recognition sites (restriction sites) for endonucleases: can have new genes inserted
- have an antibiotic resistant marker (selective marker)
- have a promoter or origin point for self replication
13
Q
making recombinant plasmids
A
- cut plasmid DNA and foreign DNA with the same endonuclease
- mix plasmids and foreign DNA
- add DNA ligase which joins the sticky ends together
- same recognition site allows same restriction enzyme to cut both the plasmid and the gene of interest
- produces matching sticky ends on the plasmids and the gene of interest
- plasmids will then join with the DNA of the gene of interest to form recombinant plasmids as they have complementary sticky ends.
- the two fragments can then be successfully joined by DNA ligase.
14
Q
recombinant plasmid
A
a plasmid that has taken in new/ foreign DNA
15
Q
bacterial transformation
A
- when bacteria cells take up plasmids
- recombinant plasmids need to be taken up by bacteria
- techniques that temporarily interfere with the plasma membrane allow this (causes holes in the pm so plasmid can enter)
- electroporation
- heat shock
16
Q
how is transformed bacteria identified
A
- bacteria can take up recombinant plasmid with AmpR gene
- results in bacteria resistant to ampicillin antibiotic
- bacteria can take up recombinant plasmid with green fluorescent protein (gfp gene_
- when uv light is used, bacteria is fluorescent
- but needs arabinose , sugar to repress the repressor of the gfp gene