Manipulating genomes Flashcards
Sequencing - Why are so many copies of the unknown DNA sequence needed?`
To ensure multiple copies of every possible length strand is produced, and no bases are ‘missed’.
Seq - What is the function of the primers
To give DNA polymerase a double stranded section of DNA to start with - a strand to elongate.
Seq - What is the function of the dNTPs
To elongate the strand.
Seq - What is the function of the ddNTPs?
To terminate the strand.
Seq - What is the role of electrophoresis in the process
To separate the strands according to length - shortest emerges from the process first.
Seq - How is the final sequence produced?
A laser causes the labelled ddNTP to fluoresce and give out a pulse of coloured light. This is detected and recorded on an electropherogram.
Seq - How are very long (»> 1000bp) sections of DNA sequenced
- Shotgun sequencing.
- Multiple copies are randomly cut and the fragments sequenced.
- Computers look for overlaps and the fragments are assembled into the original sequence
What is a genome?
all of the genes (including mitochondrial) - both the coding and non-coding DNA
Sanger seq basics?
- essentially DNA rep but using a mixture of normal nucleotides that allow the new strand to elongate and a set of chemically altered nucleotides that terminate the elongating chain
- these altered nucleotides are labelled w a fluorescent marker that can be recognised by an automatic seq. machine
dNTP?
adds to 3’ end of DNA strand, elongating the chain
ddNTP?
adds to 3’ end, terminating it
how does electrophoresis produce an electropherogram?
- As the DNA fragments emerge in the capillary tube, a laser causes the markers to fluoresce
- this is picked up by a sensor and an electropherogram is produced showing the base seq
Shotgun seq?
• sequenced fragments from sanger seq ordered using shotgun sequencing
weakness of shotgun seq?
unclear where the repeat sequences overlap if repeat seq
PCR?
Used for making many copies of a specific seq. of DNA
What’s needed for PCR?
- Original DNA sample - the template
- short DNA primer that are comp to the start and the end of the seq to be amplified - the primers ‘bracket’ the seq
- ddNTPS - all 4
- a thermostable DNA polymerase usually Taq polymerase
- thermal cycler machine
- Eppendorf tubes
3 stages of PCR?
- Denaturation
- Primer annealing
- Elongation
- Denaturation?
- 95 degrees c for 30s
* ds DNA is separated into ss by breaking H bonds between the strands
- primer annealing?
- 55 degrees
* primer bind to the start and end of the DNA template by complementary binding
- elongation?
- 72 degrees for at least 1 min
- DNAP extends the primers
- works best at 72, it’s the optimum T
- DNAP adds to bases to the primer, building complementary strands of DNA, producing a ds DNA molecule identical to the original seq
after elongation?
- then step 1 again
* repeating this process results in an exponentially inc the no. of DNA molecules
in a diagram the primers would be at ? sides of opp DNA strands?
opposite, DNA strands are antiparallel
Benefits of PCR?
+ detects mutation
+ recombine - gene therapy - attaching DNA to some other genome
+ paternity testing
+ solving crimes
Applications of PCR?
- start of sanger seq, to make copies of the DNA to be seq
- DNA profiling -many copies of the DNA sample are made before RE used
- making copies of a gene being inserted into a vector for GE
- Amplification of small DNA samples for testing e.g. pre-natal diagnosis
Electrophoresis uses?
- ss
- DNA profiling
- can be used to separate proteins too
- separates diff lengths of DNA
what moves the furthest in electrophoresis?
- Smallest DNA fragments
- less resisted by gel
- so detected first
How does shotgun seq work?
- multipole copies of the genome are fragmented randomly into short, sequencable lengths
- fragments are seq
- then overlapping base sequences are identified using computers
- the fragments can then be put in order and large seq of DNA can be assembled
new seq tech?
- inc speed of seq
- brought down costs
- allowed whole genome seq to happen
uses of seq?
- find alleles giving predisposition e.g. cancer - preventative treatment
- diagnose genetic conditions - e.g. preimplantation genetic diagnosis
- discover phylogeny/ evolutionary relationships
- adds to sci knowledge/ database of sequences
why is Taq polymerase used?
can heat to high temps, other DNA polymerase would denature
why is a thermal cycler machine used?
can change T
new high throughput techniques?
use nanopores - artificially constructed channel proteins thru which DNA moves, reading its base seq
DNA profiling basics?
• everyone has a unique combination of DNA (except identical twins)
• but is more similar to close family members
• so DNA profiling is a useful tool for:
paternity testing, forensics, classification of organisms, pre-disease
DNA profiling involves looking at sections of DNA which?
- vary between individuals
- could be STRs
- VNTRs
- specific alleles
STRs?
short seq of non-coding DNA that repeat a diff no. of times in diff indivis
VNTRs?
- minisatellites/microsatellites/ VNTRs
* repeating sections of non-coding DNA found in the introns of the genome
looking at specific alleles?
specific alleles for specific genes that are known to give e.g. a predisposition can be looked at
how are these variable regions found?
- probes (short lengths of radioactively labelled DNA)
- are made that are comp to the seq of bases of interest
- the probes bind to the DNA of interest and show up as bands on a DNA profile
DNA profiling uses?
- paternity
- forensics
- other familial relationship testing
- ancestry investigation - e.g. 23 & me