GAG WK1 Flashcards
What is a genome
all the genetic information of an organism
What are the 2 main human genomes
nuclear & mitochondrial
What are the features of nuclear genome
- found in the nucleus
- most of the organisms’ genes (larger genome)
- contains linear DNA organised into 23 pairs of chromosomes
What are the features of mitochondrial genome
- found in the mitochondria
- contains 37 genes & involved in energy production
- smaller genome
- contains circular DNA
DNA definition
a polymer of nucleotides that forms double helix
What are features of double helix structure
- they have major & minor grooves
- they can form different double helix structures that can affect its regulation
What is a feature of the major grooves of DNA
- they have more open space so they can allow proteins in
- (major/minor grooves affect protein binding)
How is the DNA Structure so stable
- base pairings with H bonding
- base stacking allows hydrophobic effect, and Van der Waals interaction maximises favourable interactions
What is DNA Packaging
DNA is wrapped around nucleosomes to help contain DNA in nucleus
What are histones
proteins associated with DNA that have tails which can influence gene expression via epigenetic modification
What are nucleosomes
they are made up of 4 pairs of histones and form an Octomer
What are the 2 main types of chromatin
- euchromatin
- heterochromatin
What are the features of euchromatin
- loosely packed + less condensed chromatin
- found in transcriptionally active regions (most chromosomes)
- accessible to transcription for active gene expression
What are the features of heterochromatin
- tightly packed + less condensed chromatin
- found in inactive genes or repetitive DNA sequences (ex: telomeres)
- non-accessible to transcription for silencing genes
What are karyograms
visual representation of chromosomes showing distinct banding patterns (organised in descending size order)
Why would karyograms be useful
- identify chromosomes
- detecting chromosomal abnormalities
What kind of genes does mitochondrial genome encode for
essential genes for mitochondrial functionn
What is DNA sequencing
determining the order of nucleotides in DNA
What is the purpose of DNA sequencing
- identify: mutations + structure + genes
What are the 3 main types of DNA sequencing
- Sanger Sequencing
- Next generation sequencing
- nanopore sequencing
What is Sanger sequencing
sequencing DNA by making DNA fragments different lengths
Outline the 5 main steps of sanger sequencing
- DNA denaturation
- Primer annealing
- Strand (DNA) Synthesis
- Chain termination
- separating DNA fragments via electrophoresis
What happens during Step 1 of Sanger sequencing
- DNA denaturation
- double strand is denatured into single strands
What happens during step 2 of Sanger sequencing
- primer annealing
- primer is added to start strand synthesis
What happens during step 3 of Sanger sequencing
- strand synthesis
- DNA polymerase extends strand w/ dNTPs
What happens during 4th step of Sanger sequencing
- Chain termination
- ddNTPs are randomly added to stop strand synthesis
- each ddNTP has a fluorescent dye
What happens during step 5 of Sanger sequencing
- separating DNA fragments via electrophoresis
- They are separated by size, and the detectors reconstruct the DNA
Why do ddNTP terminate DNA synthesis
- they lack 3-OH group and cannot make phosphodiester bonds
What are the benefits of Sanger sequencing
- they can sequence small pieces of DNA (small-scale sequencing)
What is Next Generation Sequencing
it allows millions of DNA fragments to be sequenced at once via parallel sequencing
What is parallel sequencing
it sequences DNA in parallel and not one fragment at a time
What are the 2 main immobilisation methods used in NGS
Glass slide & metallic beads
What are immobolisation methods
methods used to attach DNA fragments to a surface so that they can be fixed and used for parallel sequencing
What is nanopore sequencing
it reads DNA sequences without DNA synthesis
Outline how nanopore sequencing works
- Helicase unwinds DNA into single strands
- DNA strand moves through nanopore
- Each base changes the electrical current as it passes through
- the recoding of the electrical current indicates DNA sequence
What are the benefits of nanopore sequencing
- it is good for complex genome projects because it can sequence long reads
- it is very fast because it is real-time sequencing and provides immediate analysis
What are the limitations to nanopore sequencing
- higher errors > Sanger & NGS
- faster sequencing = reduced accuracy
What are the benefits of NGS
- cost-effective (produces larger amount at lower costs)
- rapid genome analysis (can sequence millions of DNA fragments at once)
- good for genome projects
What are the limitations of NGS
- higher error rate > Sanger sequencing
- short read lengths (compared to nanopore)
What is the human genome project
- it started in 2003
- Sanger sequencing was used to sequence the whole human genome
What were the benefits of the human genome project
- identify mutations
- improve health and prevent diseases
What were the limitations of human genome project
- lack of diversity (giving false positives in disease studies)
- it mostly consists of European ancestry
- it was the result of private and public findings
What is a gene
DNA sequence that contains information to produce a functional product made of RNA or protein
What are the 3 main types of RNA
- mRNA
- tRNA
- rRNA
What is the function of mRNA
carries genetic information to ribosome
What is the function of tRNA
carries amino acids to ribosomes
What is the function of rRNA
structural component of ribosomes
Why are genes the minority of the genome
- most of the genome consists of non-coding regions (ex: introns & repetitive sequences)
- genes are interrupted by introns (increases size of genome without adding protein-coding regions)
- a lot of the genome is involved in regulating gene expression
Name structures of a eukaryotic gene
- insulators
- promoter region
- TATA box
- transcription start site and transcription termination site
What are insulators
- they prevent regulatory regions of other genes from interfering with gene expression
- block enhancers or silencers from neighbouring genes
- act as a barrier against heteromatin spreading into euchromatin regions
What is the promoter region
- DNA sequences found upstream of the transcription start site
- have TATA box
- regulate RNA polymerase binding and transcription initiation
What is the regulatory region
they regulate gene expression (ex: enhancers or silencers)
What is the transcription start site
RNA polymerase begins transcription
What is the transcriptional termination site
it signals the end of transcription
What is transcription
the process of synthesising DNA into RNA
What are the 3 stages of transcription
- Initiation
- Elongation
- Termination
What happens during initiation stage of transcription
- RNA polymerase binds to promoter region of gene
- transcription factors guide RNA polymerase to transcriptional start site
What happens during elongation stage of transcription
RNA polymerase moves along the DNA synthesises a complementary RNA strand
What happens during termination stage of transcription
- transcription continues until it reaches a termination site
- RNA strand is released
What is the purpose of post-transcription modifications
- to increase the stability of RNA transcript
What are the 3 post-transcription modifications
- 5’ capping
- RNA splicing
- 3’ poly-A tail addition
What is 5’ capping
protects RNA transcription from degradation and allow export from nucleus
What is RNA splicing
the removal of introns to allow just protein-coding DNA/regions
What is 3’ poly-A tail
adding adenosine to add stability to mRNA and promote translation
What is translation
the process of synthesising RNA into protein
What happens during initiation of translation
- ribosomes assemble around mRNA
- start codon (AUG) on mRNA signals start of translation
- tRNA brings Met (amino acid) to start codon via its anti-codon
What happens during elongation of translation
- ribosome moves along mRNA as tRNA brings complementary amino acids
- A site (entry amino acid), P site (peptide bond formation), E site (exit of tRNA)
What happens during termination of translation
- translation terminates once the stop codon is reaches
- ribosomes disassemble and polypeptide is released
What is the function of PCR
it amplifies DNA and RNA
What is the benefit of PCR
- making copies of genes
- identify specific mutation or allele
What is the function of qPCR
quantifies the amount of DNA or RNA with fluorescent dyes or probes
What is the relationship between fluorescence and DNA (qPCR)
- the fluorescence intensity increases with each cycle = amount of DNA produced
What is the function of SDS-PAGE (Gel electrophoresis
separates proteins based on weight/size
How does SDS-PAGE work
- SDS (a detergent) and heat denatures proteins –> gives a negative charge
- the gel allows smaller proteins to move faster through the gel
- proteins migrate towards the positive electrode due to negative charge
What is Western Blotting
it uses antibodies to detect specific proteins after separation
How does Western Blotting work
- primary antibodies bind to the target protein
- Secondary antibodies bind to primary antibodies –> this amplifies the signal making it easier to detect target protein
What is the relationship between the intensity of signal in Western blotting and protein
- greater intensity of signal = more protein present
- more target protein = more primary antibodies = more secondary antibodies bind = stronger signal detected
