ביוטכנולוגיה

סיכום שיעור 10

SophieSacofsky

SophieSacofsky

פרטי הסיכום

קורס: ביוטכנולוגיה

מספר השיעור: 10

סיכום השיעור

Do trees have senses? What do they feel? They speak to other trees and speak within themselves. For example, where the light is where the tree grows towards. But we don’t think they hear.

A Venus fly trap saves energy. It only closes when the sensors are activated that there’s an insect there. Theres an electric current.

Green algae is thought to be the oldest ancestor. 75% of oxygen we breathe is connected to algae.

Glow in the dark trees – saves electricity. Can light up the road.

“guns, germs and steel” book – there were 22 types of homo. Only one survived but we weren’t the cleverest. There is strength to being social and living in a community. Once farmers were responsible for producing food, other people were able to do other jobs, focusing on other things except finding food. Armies were better so could conquer more lands.

SEQUENCING:

How much RNA expression? What DNA changes are there?

Real time PCR – corona tests, 3 primer pairs to spike.

Deep sequencing – all RNA in a few tissues (NGS).

3-5% of RNA is mRNA. Most is rRNA. Eukaryotic RNA has poly A tail. RNA is not stable, degraded easily. RNA needs to be clean. RNA needs to be checked to see if can continue protocol with it.

Microarray – oligos that we have chosen. Lots of the same probe. Put RNA/cDNA on chip. Complementary sequence attaches. There is a fluorescent signal. Its relative. Healthy vs ill. Over or under expressed. Not absolute. There is a specific amount of oligos. There is a problem of resolution. Used for gene expression, SNP, alternative splicing. Agilent vs affymetrix.

NGS/high throughput – in 1990 cost $2.7bn, now costs $1000. Increased specificity. Absolute.

Sanger – replication stopping fluorescent nucleotides. Use x ray to read.

Capillary – laser scanner to give sequence.

Novaseq – 400mn reads

Illumina – sequencing by synthesis. Nucleotide is fluorescent so read the flash, then loses fluorescence.

sample preparation – extraction to cDNA from RNA, adapters, index, poly A tail.

cluster preparation – cDNA on flow cell replicated. P5 and p7 primers. Bridge. Only F left.

Sequencing – nucleotide added and colour seen by detector. Gene then index then do bridge. R.

Analysis – millions of reads. Look at indexes. Aligned to reference genome.

Shearing – covaris (sonicator), nextera (transposomes cut and label at same time. Random cutting).

RNA library prep – use bead to attach mRNA using poly A tail. Wash remaining material.

Single cell RNA sequencing – cell surface proteins, intracellular proteins, mRNA, histone modifications, genome sequencing, DNA methylation.

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