Sanger Sequencing + reading (week 5 Thursday) - BIMM121

what does sanger sequencing need?

• DNA polymerase (enzyme)

• template DNA

• fluorescently dyed dideoxynucleotides

• primer

• nucleotides

• buffer

first step of sanger sequencing:

denature at high temp, then cooled allowing primer to bind

what happens in sanger sequencing once primer binds?

DNA polymerase adds nucleotides until a random dideoxynucleotide is placed

what is the dideoxynucleotide missing?

the hydroxyl group, which is what usually allows the next nucleotide to be added. so this terminates the chain

what does the fluorescent color represent on the dideoxynucleotide?

the base that was added

how is sanger sequencing read?

each time a dideoxynucleotide is terminated, the chain is read in order from shortest to longest and the fluorescent dyes are used to read what base is at each location

The Human Genome Project

took 13 years and thousands of researchers, helped scientists w genetics of cancer and diseases while creating at home DNA testing

Illumina recently revealed what?

it’s fastest machine yet at whole genome sequencing called NovaSeq X series

sequencing has led to what?

genetically targeted drugs, blood tests that can detect cancer early, and diagnoses for people with rare diseases

how long did the human genome project take?

13 years and thousands of researchers

final cost of the human genome project:

$2.7 billion

the 1990 project kicked off what?

the age of genomics

what did the project help with?

helped scientists unravel the genetic drivers of cancer and many inherited diseases while spurring the development of at-home DNA tests

how much does genome sequencing cost?

10 years ago: $10k

a few years ago: $1000

now: $600

Illumina controls how much of the DNA sequencing market globally?

80%

how much will these machines make sequencing cost?

$200

how many sequences a year can the machines do?

currently 7500, aiming for 20k genomes a year w the more powerful model

what has sequencing lead to?

• genetically targeted drugs

• blood tests that can detect cancer early

• diagnoses for people w rare diseases

• Covid-19 vaccines

why is sequencing still unaffordable (why its not ubiquitous in medicine yet)?

while it costs $600 for the sequencing alone, clinical interpretation and genetic counseling can drive the price into the thousands

another reasoning sequencing isn’t ubiquitous in medicine yet:

for healthy individuals genome sequencing doesn’t really help with anything, although in 12-15% of healthy individuals, genetic variations that are linked to increased risk of disease were caught

right now who benefits most from sequencing?

researchers not patients

problem with current genetic data sets?

genomes that have been sequenced come from mostly people of european descent, different races could have different disease-causing genetic variaton

what method do Illumina’s sequencers use?

sequencing by synthesis

• split double-stranded DNA into single stranded

• dna broken into short segments and spread on a flow cell

• when flow cell is added to macinery, fluorescent colored tags attach themselves to the nucleotides

• each of the DNA fragments copied one base at a time

• matching strand of DNA made

• laser scans the bases one at a time while camera records the color coding for each letter

How has Illumina improved its latest machines?

• denser flow cells

• can resist heat, water

• much tougher, more laser power and scan them quicker

machines still have a steep cost…

$1 million, but don’t need team of scientists because machine does analysis in comparison to already sequenced genomes

what is Illuminas largest competitor?

Ultima

snager sequencing advantages:

• cost effective in small batches

• highly accurate

• larger read >500bp

sanger sequencing disadvantages:

• only analyze one DNA fragment at a time

• expensive for large scale sequencing

• not effective at genome sequencing

• 700-100bp