rDNA technology and genomics Flashcards
How can DNA be prepared for studying?
- homogenise cells or tissues
- centrifuge.. the aqueous layer is nucleic acids and the phenol layer is protein
- addition o ethanol and salt causes the nucleic acids to precipitate out of solution
How are type II restriction endonucleases used in DNA extraction?
- cut from within the DNA to cleave specific sequences
- usually cleave palindrome sequences
- cuts both strands of DNA causing them to separate
- the sticky ends are then left unpaired with DNA (overhang)
How can compatible DNA overhangs be re-joined?
When the DNA is cleaved it can produce compatible overhangs that when combined are no longer recognised by the endonuclease.
When they are rejoined a gap remains that is rejoined by DNA ligase
What is agarose gel electrophoresis?
- separates DNA fragments by size and charge
- DNA moves towards a positive electrode at varying speeds based on its size
- its exact size is then determined using a mwt marker made of fragments of known sites
What is recombinant DNA?
an artificial DNA sequence generated by combining DNA from multiple organisms
What is gene cloning?
Isolating and making identical copies of a fragment of DNA
What are DNA carries/vectors
vectors that carry and replicate foreign DNA fragments within a cell
What are the 5 steps of gene cloning?
- isolate bulk DNA
- isolate specific DNA sequence
- ligation (joining DNA fragment with a self-replicating genetic element
- transform bacteria (force into vector)
- screen bacteria
What do vectors do?
Carry and propagate DNA in host cells by autonomous replication
How is bacteriophage lambda used as a vector?
- contains a region of DNA that is not required for survival or replication
- this can be taken and replaced by foreign DNA
- when lambda lyses within a bacterium it will released particles each carrying a copy of the foreign DNA
What is a bacterial circular plasmid vector?
Supercoiled, double-stranded DNA circles that exist independent of the bacterial genome and can carry foreign DNA
How is a vector joined with foreign DNA?
- restriction enzyme creates a sticky 5’ overhang
- the foreign DNA is cleaved with the same enzyme to produce a compatible sticky overhang
- these reanneal with DNA ligase and a recombinant plasmid is formed
How is the recombinant plasmid introduced into bacteria?
- treat the bacteria with CaCl2 and heat to make them leaky
- Those that take up the plasmic will now have antibacterial properties so the bacteria can be treated with ampicillin to remove those that haven’t taken it up
How are eukaryotic genes cloned?
- reverse transcriptase synthesised cDNA using a primer that binds the poly(A) tail of mRNA
- RNAse H digests the mRNA to form a single strand of DNA
- the ssDNA forms a hairpin that primes DNA strand synthesis and DNA polymerase is added to make dsDNA
Why can mature mRNA NOT be used for cloning?
It is not double stranded
What is the role of the polymerase chain reaction?
- allows selective synthesis of a gene sequence using prior knowledge of the gene sequence to design ssDNA primers
- PCR amplification Is exponential
What is taq DNA polymerase
- DNAp with enhanced stability at high temperatures
- can withstand repeated heating and cooling without denaturing
- lacks 3’-5’ proof reading
What is the Tm?
The temperature where 50% of primer is annealed to the template
What happens if the primer anealing temperature is incorrect?
- too high = no annealing, no product
- temp too low = mismatches can occur and non-specific products are created
What are the 3 parts of the PCR amplification cycle?
denaturation, primer annealing and extension
What happens at 95 degrees, 60 degrees and 70degrees in the PCR amplification cycle?
95 - heat denatures dsDNA h-bonds to make ssDNA templates
60 - primers anneal
72 - optimal temperature of taq polymerase DNA synthesis
How many times is the PCR amplification cycle repeated?
25-35 times
What are the long product and desired produced in PCR?
Long = doesn't have the required 3' end as nothing stops the primers from synthesising along the entire strand Desires = in the next PCR step, the products from step 1 hybridize to the strands and help to form a defined end.
Name three uses of PCR?
- gene cloning
- viral screening
- forensics/DNA fingerprinting
What is DNA sequencing?
- determines the order of bases in a DNA sequence
- can screen of gene mutations and sequence variants
What is sanger sequencing?
- uses di-deoxyribonucleotides to obtain the sequence
- no 3’OH group means it can no longer form a phosphodiester bond and synthesis ends
What is sanger DNA sequencing using ddNTPS? (use CTP as an example)
- The primer binds and synthesises DNA
- Whenever a G is encountered, either a dCTP or a ddCTP is incorporated
- when a ddCTP is selected, chain termination occurs
- this reaction can be set up for each dNTP and the sequence can be found by where the chain is stopped each time
What is bioinformatics?
The combination of computer science, statistics and maths to analyse biological data
What is comparative genomics? Give examples.
- comparing whole genome sequence of different organisms
- human/chimp
- human brain evolution
What are SNPs?
- any base variation between 2 or more individuals
- can be inherited in blocks known as haplotypes
What do genome-wide association studies on haplotypes show?
- most haplotypes will segregate randomly but some will be found more frequently in individuals with specific diseases
