Lecture 14 - Genomes & Karyotypes

Question 1

methods for studying genomes:

Answer 1

(1) genome sequencing

(2) snp arrays

(3) karyotypic analysis

Question 2

two types of DNA sequencing:

Answer 2

small scale (gene/plasmid)

large scale (whole genome)

Question 3

what is sanger sequencing?

Answer 3

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

Question 4

what type of sequencing did the human genome project use?

Answer 4

the human genome project used sanger sequencing (this would’ve taken forever - very labour intensive!)

Question 5

what is next generation sequencing?

Answer 5

they are all “high throughput” sequencing methods (or massively parallel)

Question 6

how does next generation sequencing broadly work?

Answer 6

(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

Question 7

what NGS sequencing do we look at at sussex?

Answer 7

illumina

Question 8

next generation sequencing advantages and disadvantages:

Answer 8

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)

Question 9

limitation of short read NGS sequencing:

Answer 9

• can fail to detects larger structural variants and to accurately sequence highly repetitive regions of the genome

Question 10

what form of sequencing is Nanopore sequencing?

Answer 10

Nanopore sequencing is long-read sequencing <2,000,000bp

Question 11

how many bases travel through a nanopore in a given second?

Answer 11

400 bases per second

Question 12

advantages and disadvantages of nanopore:

Answer 12

advantages:
- ultra long read lengths
- very inexpensive (few grand)
- small, portable device

disadvantages:
- less accurate at bp level (particularly AAAA/TTTTT/GGGG/CCCC)

Question 13

SNP =

Answer 13

single nucleotide polymorphisms

Question 14

how do we detect SNPs?

Answer 14

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

Question 15

what are GWAS arrays really good for?

Answer 15

during ‘gene discovery’ where a population are tested to see if any SNP variants give immunity etc to a certain disease

Question 16

what can SNP arrays be used for in a diagnostic setting?

Answer 16

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

Question 17

what do array comparative genome hybridisations (array CGH) allow for?

Answer 17

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

Question 18

using cytogenetics to detect chromosomal abnormalities:

Answer 18

using “chromosome painting” to make the chromosomes glow

Question 19

cloning plasmid vectors:

Answer 19

contain replication origin, selectable marker and a multiple cloning site MCS

Question 20

PCR PRIMER

Answer 20

16-20long

extended 5’ > 3’

annealing temp 50-60C