Lecture 14 - Genomes & Karyotypes Flashcards
methods for studying genomes:
(1) genome sequencing
(2) snp arrays
(3) karyotypic analysis
two types of DNA sequencing:
small scale (gene/plasmid)
large scale (whole genome)
what is sanger sequencing?
a type of small scale sequencing which uses Di-deoxy Nucleotide TriPhosphates (ddNTPs) that can’t form phosphodiester bonds which are chain terminating
this allows you to create a series of fragments corresponding to every base in your DNA sequence (many different sizes)
the colour of each fragment tells you the base at that position
what type of sequencing did the human genome project use?
the human genome project used sanger sequencing (this would’ve taken forever - very labour intensive!)
what is next generation sequencing?
they are all “high throughput” sequencing methods (or massively parallel)
how does next generation sequencing broadly work?
(1) extract DNA (e.g: tumour from newborn etc)
(2) library preparation: fragment DNA into short fragment (few 100 base pairs) and ligase them to short “adapter” sequences
(3) apply this to the NGS machine and all the millions of sequence reads are computationally analysed for your final product
what NGS sequencing do we look at at sussex?
illumina
next generation sequencing advantages and disadvantages:
advantages:
- high thoroughput/massively parallel (1.5x10^12 bp/run)
- high accuracy >99%
disadvantages:
- high cost of sequencing machines and reagents
- short read lengths (300bp)
limitation of short read NGS sequencing:
- can fail to detects larger structural variants and to accurately sequence highly repetitive regions of the genome
what form of sequencing is Nanopore sequencing?
Nanopore sequencing is long-read sequencing <2,000,000bp
how many bases travel through a nanopore in a given second?
400 bases per second
advantages and disadvantages of nanopore:
advantages:
- ultra long read lengths
- very inexpensive (few grand)
- small, portable device
disadvantages:
- less accurate at bp level (particularly AAAA/TTTTT/GGGG/CCCC)
SNP =
single nucleotide polymorphisms
how do we detect SNPs?
using SNP arrays using tiny beads within a well all of which are attached to a different oligo, the beads will either go red or green or both to show what SNP is/isnt present
what are GWAS arrays really good for?
during ‘gene discovery’ where a population are tested to see if any SNP variants give immunity etc to a certain disease
what can SNP arrays be used for in a diagnostic setting?
SNP arrays can be used in IVF to test which embryos do/don’t have the disease gene so that the healthy embryos can be selected
what do array comparative genome hybridisations (array CGH) allow for?
allows you to see the losses and gains of chromosomes by comparing your sample to a reference
you mix your patient and reference sample and hybridise them, where you see a green spot it means there’s more copies in the patient than in the reference and when you see a red spot you have more copies in the reference genome than patient
green = patient extra chromosomes
red = patient missing chromosomes
using cytogenetics to detect chromosomal abnormalities:
using “chromosome painting” to make the chromosomes glow
cloning plasmid vectors:
contain replication origin, selectable marker and a multiple cloning site MCS
PCR PRIMER
16-20long
extended 5’ > 3’
annealing temp 50-60C
