Summary
Summary Molecular Biology of the Cell
- Course
- Institution
Summary of the lectures of Molecular Biology of the Cell
[Show more]
Seller
Follow
liezemies
Reviews received
Content preview
Molecular Biology of the CellLecture 1 (06/09/2022) – Replication, sequencing & PCR
The central dogma → replication-transcription-translation
DNA → RNA → protein → metabolite → phenotype
DNA replication → semi-conservative
➔ Every new double stranded molecule
consists of one old and one new strand
➔ Old strand → template for new strand
DNA synthesis → the process of formation of phosphodiester bonds
while hydrolyzing the matching dNTP molecule
- Direction of synthesis is 5’ end to 3’ end due to the OH-group
DNA polymerase → synthesizes DNA from a double stranded “primer”
Replisome → molecular machine, consists of multiple proteins, all involved in
replicating the DNA strands
Mistakes in DNA synthesis → can be restored through
proof-reading activity of the DNA polymerase complex
➔ Frequency of mistakes:
o without proof-reading → 1 error per 105 NTs
o with proof-reading → 1 error per 107 NTs
o with strand-directed
mismatch repair → 1 error per
1010 NTs
5’→3’ polymerization
3’→5’ exonucleolytic proofreading
Chemical changes in DNA bases can cause mutations
- Depurination → loss of purine base (A or G)
- Deamination → loss of amino group (C→U)
➔ Point mutations or deletions
,DNA sequencing → determine the order of the bases A, C, G, and T
PCR → Polymerase Chain Reaction → DNA amplification
Dideoxy sequencing (Sanger) → DNA synthesis while incorporating chain terminators in separate reactions
➔ Chain terminators (dideoxynucleotides) → lack the 3’-OH necessary for strand extension
- With a high concentration of all dNTPs and low
concentrations of ddATP (ddCTP , ddGTP, ddTTP) in
separate reactions DNA synthesis will continue, but
occasionally synthesis will stop when a dideoxy
nucleotide is incorporated
- With four separate reactions and a labelled primer,
fragments of different lengths are generated →
separated alongside electrophoresis every visible
fragment represents a termination in DNA synthesis
Genome sequencing → BAC libraries and “shotgun” fragment sequencing
➔ Assembly by comparison of overlapping sequences
Shotgun sequencing
Contigs → assembly of smaller DNA sequences into one continuous
The “innovation” of massive parallel sequencing → reactions take place in
picolitre volume on microscopic beads
Sequencing technique → pyrosequencing
➔ Incorporation of a dNTP
molecule supplies PPi that is
converted to ATP. Every ATP
molecule is consumed by luciferase
to yield 1 flash of light
Same principle with fluorescence →
Ion torrent sequencing → incorporation of a nucleotide results in release
of a proton. Resulting pH change can be measured on a chip
Massive parallel sequencing → differences between techniques
- Bulk DNA sequence data
- Read length
- Error rate
Whole genome sequencing is used for reconstruction of genomes of
extinct species like cave bears, mammoth, neanderthal
, Polymerase
Chain Reaction
(PCR) →
amplification of
1 specific DNA
fragment
PCR → cloning genomic fragments
- Knowledge required → homologous DNA
sequence & amino acid sequence protein
- Advantages → sensitive & fast
- Restrictions → fragment length (< 20kb) &
homologous DNA sequences
RT-qPCR → cloning of specific cDNA fragments
Techniques for detecting DNA polymorphisms
- PCR-BASED → VNTR (Variable
Number Tandem Repeats)
- PCR and sequencing-
BASED/melting curve → SNP (Single
Nucleotide Polymorphism)
, Lecture 2 (06/09/2022) – Genomes, omics & bioinformatics
Bioinformatics → how to handle the enormous amount of information produced by DNA sequencing, RNA
expression, protein patterns, metabolite contents, digitalized phenotypes
There is no correlation between genome size and organismal complexity → amoeba has a greater genome
Only 2% of the human genome codes for proteins
Repeated DNA sequences → important for regulation of gene
expression and maintaining DNA structure
Genomics
➔ DNA sequence analysis, genome fragments
➔ DNA database (NCBI, Genbank, EMBL)
➔ DNA markers (SNPs etc.)
Utilization of databases:
- Compare unknown fragments
- Identification gene fragments (annotation)
- Chromosome structure/synteny
- Intron/exon boundaries
- Phylogeny
Chromosomes contain many duplicated segments
→ intra and inter-chromosomal duplications
Genome annotation → process of identifying the
locations of genes and all of the coding regions in
a genome and determining what those genes do.
Once a genome is sequenced, it needs to be
annotated to make sense of it
Exon length is conserved between human, fly and worm → suggests functional restriction splicing machinery
Intron length is much more variable in human, peaking at 87 bp, but trailing till 3,300 bp...
➔ Suggests that exons (not introns) might have a limit in size in order to splice the mRNA
Alternative splicing → splicing on places that you did not predict (not on normal splice sites)
- 35% of the genes have alternative splicing
- 70% in the coding sequence → alters the protein
- 20% terminal exon added
Differential splicing → one gene produces different mRNAs that
code for different proteins
Synteny → preserved order of genes between related organisms
- Since the order of genes mostly has a neutral effect in
eukaryotes, an organism will have no ill effects from having genes re-arranged
- The order of genes is generally preserved best between tightly related species → conservation of the
order of a cluster of genes suggests a functional relation
Synteny helps you formulate a hypothesis for gene function
Natural selection → changes in DNA that do or do not affect the encoded protein
- Ka = non-synonymous substitution ratio → base change leads to different amino acid
- Ks = synonymous substitution ratio → base change leads to same amino acid
o Ka/Ks<1 strong selection; Ka/Ks>1 NO selection
The benefits of buying summaries with Stuvia:
Guaranteed quality through customer reviews
Stuvia customers have reviewed more than 700,000 summaries. This how you know that you are buying the best documents.
Quick and easy check-out
You can quickly pay through credit card or Stuvia-credit for the summaries. There is no membership needed.
Focus on what matters
Your fellow students write the study notes themselves, which is why the documents are always reliable and up-to-date. This ensures you quickly get to the core!
Frequently asked questions
What do I get when I buy this document?
You get a PDF, available immediately after your purchase. The purchased document is accessible anytime, anywhere and indefinitely through your profile.
Satisfaction guarantee: how does it work?
Our satisfaction guarantee ensures that you always find a study document that suits you well. You fill out a form, and our customer service team takes care of the rest.
Who am I buying these notes from?
Stuvia is a marketplace, so you are not buying this document from us, but from seller liezemies. Stuvia facilitates payment to the seller.
Will I be stuck with a subscription?
No, you only buy these notes for $11.41. You're not tied to anything after your purchase.
Can Stuvia be trusted?
4.6 stars on Google & Trustpilot (+1000 reviews)
67096 documents were sold in the last 30 days
Founded in 2010, the go-to place to buy study notes for 14 years now