GEN 3: Defining the Genome II - DNA Flashcards
1
Q
List some methods used to analyse DNA and when they were first developed
A
- Sanger Sequencing: 1970s
- Restriction enzymes: 1970s
- DNA cloning with Restriction Enzymes: 1972
- Southern Blotting: 1975
- Polymerase Chain Reaction: 1985
- Next generation sequencing (NGS): 2006
2
Q
Describe how Sanger sequencing works
A
- Sanger sequencing requires:
- the target DNA
- an oligonucleotide primer of ~20nt complementary to part of that DNA
- a DNA polymerase
- extends the primer, using the target DNA as template until ddNTP is added and terminates extension
- a mixture of deoxyribonucleotide triphosphates (dNTPs) and dideoxynucleotide triphosphates (ddNTPs)
- the ddNTPs lack the 3’-OH group required for nucleotide chain extension
- fragments are separated by polyacrylamide gel electrophoresis or capillary electrophoresis, which distinguishes fragments differing in length by only one nucleotide
- labelling ddATP, ddCTP, ddGTP, ddTTP with different fluorophere produces coloured peaks that provide a direct read of nucleotide sequences up to ~1000 nucleotides long
3
Q
Describe how restriction enzymes work to analyse DNA
A
- restriction enzymes are endonuclease enzymes that cut double-stranded DNA at specific sequences
- they cleave phosphodiester bonds to leave free terminal 3’-OH and 5’-phosphate groups
- they are able to cleve internal bonds and circular DNA, unlike exonucleases which only cleave bonds at DNA ends
- these enzymes usually recognise short target sequences of 4 to 8 base pairs
- they can cut DNA into smaller fragments by targeting their specific restriction sites
- there are two types of restriction digestion
- restriction enzymes come from bacteria and is named after the species of bacteria from which it derives
4
Q
Describe how DNA cloning with restriction enzymes works
A
- DNA is inserted into a plasmid
- to do this, both the DNA and plasmid are digested with the same restriction enzymes
- this produces complementary sticky ends
- DNA ligase then ligates the two together
- the resulting recombinant plasmid is then introduced bacteria to generate a single colony (or clone) of bacterial cells, each carrying the same recombinant plasmid
- this is called recombinant DNA cloning or molecular cloning
5
Q
What was DNA cloned with restriction enzymes used to create?
A
- it was used to create a genomic DNA library
- genomic DNA was fragmented into millions of small pieces
- these were ligated into a plasmid vector and introduced into bacteria, so that each individual clone carried a different genomic DNA fragment
- Sanger sequencing of each member was then used to assemble the sequence of the whole genome
6
Q
Describe how Southern blotting analysed DNA
A
- mixtures of DNA fragments are separated by electrophoresis through an agarose gel and blotted onto a nylon membrane
- a specific sequence in the mixture can then be detected using a DNA probe that is radioactively or fluorescently labelled
7
Q
Using sickle cell disease, describe how Southern blotting is used to help determine the status of the beta-globin gene
A
8
Q
Describe how PCR works as a way to analyse DNA
A
- a pair of oligonucleotide primers is designed that flank the region to be amplified and are complementart tro opposite strands
- reactions contain template DNA, the chosen primer pair, dNTPs and a thermostable DNA polymerase (Taq polymerase)
- using a programmable temperature block, the PCR reaction is taken through multiple cycles of temperature incubations
- see image for why PCR is used
9
Q
What’s the amplification factor in PCR?
A
- in principle, the target sequence is duplicated during each PCR cycle
- if the PCR runs for n cycles, this results in an amplification factor of 2n
- this doesn’t happen in practice, but it is still powerful
10
Q
What is Next Generation Sequencing (NGS)?
A
- NGS methods can sequence million of short DNA fragments simultaneously
- known as massively parallel sequencing
- without the need for individual fragment isolation
11
Q
Describe how Illumina, an NGS method, works
A
- the DNA is first broken into short (<250nt) fragments that are tagged and hybridised onto oligonucleotides attached to a solid support called a flow cell
- the bound fragments are PCR amplified in situ (bridge amplification)
- generating millions of distinct clusters, each derived from a single fragment
- clusters are then sequenced in parallel (at the same time) by primer-extension, one nucleotide at a time, using dNTPs that are reversibly modified with 3’-end blocks and fluorescence tags
- after the addition of the first nucleotide, the flow cell is laser-scanned to measure the position and colour of each cluster
- the information is then stored digitally
- the flow cell is then treated to remove the fluorescent tags and 3’-end blocking groups on the newly extended primers
- this process is then repeated enough times to generate sequences reads for each cluster
- bioinformatics software is then used to compare sequence reads, to identify any overlaps and so assemble the sequence of the starting DNA
12
Q
What are some applications of NGS?
A
- whole genome sequencing (WGS):
- allows sequence variation between individuals to be compared
- transcriptome sequencing (RNA-seq):
- sequencing of DNA reverse transcribed from RNA transcripts
- the most highly expressed genes give the greatest number of sequence ‘reads’
- targeted sequencing:
- a small region of the genome is sequenced in samples where there may be many variants
- e.g. exome sequencing may reveal protein-coding variations
- ChIPseq:
- antibody to a protein of interest is used to purify chromatin containing that protein, prior to WGS
- reveals protein-genome interactions
13
Q
Referring to the illustration for Sanger sequencing, why does the incorporation of the first ddNTP (ddGTP) not prevent any further primer extension?
A
- the reaction contains many primed templates as well as a mixture of dNTPs and ddNTPs (the former being excess)
- at each position in the sequence, therefore, only a small proportion of the primers have their 3’ ends blocked
- the majority will have unblocked 3’ ends and so will be extended
14
Q
It is important to optimise the temperature at step 2 of PCR (annealing the primer)
Can you predict the consequences of step 2 temperatures that are
a) too high
b) too low ?
A
a) too high:
- primer hybridisation would be impaired so no PCR products would be obtained
b) too low:
- primers would bind with less specificity and may give rise to spurious PCR products
15
Q
At each sequencing step during Illumina NGS, what must happen in between laster scanning of the flowcell and addition of the next nucleotide?
A
- the 3’-blocks and fluorescent tags must be removed
16
Q
How many nucleotides is the human haploid genome composed of?
A
- 3 billion nucleotides
17
Q
What percentage of the human haploid genome are genes and gene-related DNA?
How many protein-coding genes are there?
What are some gene-related DNA examples?
A
- 37.5%
- there are about 21,000 protein-coding genes
- most of their DNA is non-coding
- introns, UTRs
- non-protein-coding-genes make RNAs with known and unknown functions
- rRNA, tRNA, miRNA, some lncRNAs
- other non-coding DNA is known to be gene-related but lacking function
- pseudogenes
- gene fragments
18
Q
How much of the human genome is made up of highly repeated DNA?
What is it made up of?
A
- 54%
- 1740Mb
- it is made up of dispersed transposable elements and tandemly repeated DNA
19
Q
Observe this diagram for the breakdown of the human genome constituents
A
20
Q
How is most of the DNA in protein-coding gene not coding?
A
- there are non-coding regions such as:
- 5’ and 3’ untranslated regions (UTRs)
- enhancer sequences
- promoter sequences
- long introns
21
Q
What is alternative splicing?
A
- alternative splicing allows a single gene to generate multiple mRNA isoforms
- and therefore, multiple protein isoforms
22
Q
Describe the gene density across humans, prokaryotes, simple eukaryotes
A
- human genomes have very low gene density compared to genomes of prokaryotes and simpler eukaryotes
- genes are tightly packed in bacteria and yeast (with only the occasional intron in yeast) while genes in higher eukaryotes are less tightly packed and routinely interrupted with introns
27
Q
Describe ribosomal RNA genes
- what do they code for
- transcribed by what
A
- ribosomal RNA is essential during protein translation by ribosomes
- rRNA genes are the best characterised of the ncRNAs
There are 4 rRNAs:
- 18S
- 5.8S
- 28S
- 5S
- The genes for these exist in multiple copies to ensure there is sufficient rRNA for translation
- 18S, 5.8S and 28S rRNA are derived from a 41S precursor transcribed by RNA polymerase I from ~ 300 copies of a gene that is tandemly repeated in 5 clusters on different chromosomes
- Similar numbers of 5S rRNA genes, also clustered as tandem repeats, are located elsewhere in the genome and transcribed by RNA polymerase III.
28
Q
Describe what transfer RNA genes do
Transcribed by what?
A
- codes for transfer RNA (tRNA)
- Transfer RNA (tRNA) also functions during translation by delivering amino acids to the ribosome.
- tRNAs are small; 76-90 nucleotides, with a folded structure.
- They can base pair with the codons of an mRNA strand, and this process delivers the attached amino acid to the growing peptide chain
- Genes for the 49 different tRNAs also exist as multiple copies at various chromosome sites and are also transcribed by RNA polymerase III.
30
Q
Describe micro RNA genes
What are miRNAs?
A
- MicroRNAs (miRNAs) are small (~22nt) RNAs that control gene expression by RNA interference
- they physically block translation by binding to mRNAs to prevent ribosomal access
- Many different miRNAs, each targeting specific mRNAs, are transcribed from multiple genes by RNA polymerases II or III.
- Piwi interacting RNAs (piRNAs) also work by RNA interference but are expressed only in the germ line where they silence transposons.
- The number of gene assigned to these categories is growing.
34
Q
Look and learn the example of non-processed pseudogene at the human beta-globin gene on chromosome 11
A
35
Q
What are the two types of highly repeated DNA?
A
- dispersed (or interspersed) repeats
- tandem repeats
39
Q
Describe the difference between RNA transposons and DNA transposons
Describe their mechanisms
A
- RNA transposons (retrotransposons):
- transcribe their DNA into RNA and use a reverse transcriptase to convert this back into DNA that inserts into a new site in the genome
- Remarkably, RNA transposons account for about 40% of the human genome!
- DNA transposons:
- make up about 3% of the human genome
- use a transposase to excise their DNA from one site and insert it elsewhere in the genome
- image caption: DNA transposons leave their original site to integrate elsewhere (‘cut and paste’), whereas RNA transposons remain at their original site but make copies that insert elsewhere (‘copy and paste’).
40
Q
What are the three main types of RNA transposons?
A
- LTR retrotransposons
- LINEs
- SINEs
41
Q
Use this diagram to describe how DNA transposons work
A
- DNA transposons consist of a transposes gene flanked by inverted repeat (IR) sequences
- the transposase protein recognises and cleaves the IR sequences, releasing the transposon DNA
- the transposase also catalyses the integration of the DNA at new, non-random sites in the host cell genome
42
Q
Describe LTR retrotransposons
A
- long terminal repeat (LTR) retro-transposons are also called endogenous retroviruses (ERVs)
- their DNA consist of a coding region, flanked by a pair of LTR sequences (100 bp - 5 Kb in length)
- LTR retrotransposition involves an integration mechanism related to that used by retroviruses, which also have LTR sequences
- the genomes of LTR retro-transposons include two genes:
- gag: which encodes a protein needed to make a cytoplasmic virus-like particles
- pol: codes for reverse transcriptase
- unlike retroviruses, LTR retro-transposons are non-infective, as they lack the env gene that retroviruses need to leave their host cells and infect other cells
43
Q
Describe LINEs RNA transposons
- length
- abundance
- structure
A
- long interspersed nuclear elements (LINEs) are around 6 Kb in length
- their mRNA codes for a reverse transcriptase that can copy the RNA back into DNA which can then integrate into a new site in the genome
- reverse transcription is often incomplete leading to a truncated, non-functional product being inserted back into the genome
- among the different families of human LINEs, one called LINE- 1 or L1 is very abundant
- it accounts for 17% of the human genome!
- structure:
- 5’ UTR: includes a strong promoter driving transcriptional initiation by RNA polymerase II
- Open Reading Frame 1: encodes an RNA binding protein required for transposition
- Open Reading Frame 2: encodes a protein with endonuclease (EN) and Reverse transcriptase (RT) acti ity required for transposition
44
Q
Describe SINEs RNA transposons
A
- they are short interspersed nuclear elements
- 100-400 bp
- they are non-autonomous transposons, as they do no code for any proteins
- they depend on proteins (e.g. those encoded by other transposons) for transposition
- among the different families of human SINEs, one called Alu is very abundant
- it accounts for 11% of the human genome
46
Q
What are tandem repeats?
A
- regions of DNA where an array of identical, or highly similar, repeat motifs (also called repeat units) is consecutively repeated
- less abundant than transposons
48
Q
How are tandem repeats classified?
A
- according to their abundance, size of their motifs and arrays, they are classified as:
- macro-satellites
- mini-satellites
- micro-satellites
49
Q
Describe macro-satellites in eukaryotes
- motif length
- array size
- locations
A
- they have motifs up to ~220 nt long
- form large arrays (~ 20,000 to 5,000,000 nt long) at 1-100 locations in the genome
- sequencing such large arrays is an ongoing challange and why the full sequence of the human genome sequence is still incomplete
- located in heterochromatin and at or near centromeres and telomeres
- the major human centromeric macro-satellites DNA, alpha-satellite DNA repeat motif 171 nt, is implicated in centromere function and chromosome segregation
- see diagram
50
Q
Describe mini-satellites in eukaryotes
- motif size
- array size
- location
- uses
A
- motifs of 10-150 nt
- forming arrays of ~20 - 2000 nt at more than 1000 locations in the genome
- mostly in euchromatin at telomeres and centromeres
- these regions have no clear function
- but in 1984 Alec Jeffreys famously identified that the number of repeat units in mini-satellites varies between individuals.
- This discovery led to DNA fingerprinting.
52
Q
Describe how Huntington’s disease is caused
A
- Many micro-satellites are located close to or within genes and some may even be part of the protein-coding region.
- A famous example is the tri-nucleotide repeat (CAG)n array found in the gene HTT.
- This codes for the protein Huntingtin, implicated in Huntington’s disease.
- Arrays in healthy individuals contain between 6 and 35 CAG repeats.
- Individuals affected with Huntington’s disease, however, have >35 repeats, which cause the protein product to have harmful properties.
