Describe DNA Mismatch Repair
DNA repair is absolutely faithful (repaired strand matches original strand)
1. Excision
- damaged DNA base pair is cut out by series of nucleases
- Resynthesis
- repair DNA polymerase fills gap with a new DNA base pair - Ligation
- DNA ligase seals nick left in sugar phoshphate backbone
- Nick sealing, remakes broken phosphodiester bond between adjacent nucleotides
Describe homologous Recombination
When does this occur?
shortly after DNA has been replicated but before cell has divided
- Digest
Nuclease digests 5’ ends of broken strands at the nick - Invade
Enzymes allow for 3’ broken end to invade unbroken homologous DNA to search for complementary sequence via base pair - Elongate
Repair DNA polymerase elongates invading strand (uses complementary strand as template) - Rejoin
After repair polymerase passes point where break occured:
- nwely elongated strands of rejoin og partner to form base pairs - Synthesis:
Additional DNA synthesis at 3’ ends of both strands is followed up by ligation
DNA repair is reasonably faithful:
- closely matches original sequence on both strands
- might have slight differences (allelic differences = polymorphisms)
Describe Non Homologous End Joining
- Clean
Nuclease chews back broken ends to produce flush ends (deletion of DNA sequence / loss of nucleotides) - Stitch
flush ends stitched together by ligase
Unfaithful DNA repair
Compare and Contrast the mechanisms of mutation within a gene and within regulatory DNA
Mutation within a gene:
- alter splicing of gene’s RNA transcript
- alter stability, activity, location , interaction of encoded protein / RNA product
Ex. Point mutation:
- caused by failures in copying and repairing DNA mechanisms
- low frequency of occurance (mismatch repair is effective 99% of the time)
- often has no effect on gene function (conservative sequence change does not affect amino acid sequence or occurs in non-essential portion of amino acid sequence or occurs in intron)
- rare to destroy gene activity; even more rare to improve gene activity
Regulatory Regions:
- can change expression of gene in population (if its advantageous)
ex. Lactose intolerance adults but now most of the world is lactose tolerant
Describe what is an MEI and its function
Mobile Element Insertion
Transposition of MEI:
- specialized DNA sequences move from one chromosomal location to another
(recombines due to base pairing with similar short DNA target sequences present in both chromosomes)
- results in change in activity / regulation of gene
- promote gene duplication, exon shuffling and genome rearrangements
- alter expression pattern when inserted into regulatory DNA
Describe the mechanism of Unequal Pseudo-homologous Recombination ‘Crossovers’
MEI allow homologous chromosomes to misalign –> formation of unequal template strands
- results in duplication of same gene on one chromosome
- duplicated genes undergo conservative mutational changes –> generates different but related genes (gene family)
Ex. Globin Gene family
1. ancestral globin gene was duplicated to create alpha and beta globin genes)
2. Further duplications generated sub family of beta globin (continues to branch out as more mutations arise)
What happens during Exon Shuffling
Proteins are made up of domains.
Domains are coded by usually one exon.
Thus:
- exon shuffling and gene duplication create different combinations of exons
- each combination of exons codes for a unique protein with a distinct set of domains.
Diversity of human protein strucutre and function is generated by shuffling of exons in various combinations to generate unique functions for each gene
Transposable MEI drives Exon Shuffling
- two MEis of same type insert near each other in chromosome
- transposition mechanisms recognizes the ends of 2 diff elements and causes the DNA between the two MEIs to be excised into a new site.
Describe how retroviruses interact with flow of genetic information
Retroviruses reverse normal flow of genetic information
- carry RNA as genetic material which they convert into DNA when infecting a host cell.
1. Reverse transcriptase enzyme (encoded by viral Genome and packaged with RNA) makes single-stranded DNA copy
2. creates second DNA strand to generate double-stranded DNA
3. DNA double heliz integrates with host chromosome –> allows for synthesis of viral RNA by host-cell RNA polymerase
How to treat retroviruses
Block Viral Cycle
- entry inhibitors (prevents ligand / receptor interaction )
- Inhibitors of reverse transcriptase (prevents viral DNA production)
- Integrase inhibitors (prevents viral RNA production)
- Protease inhibitors (prevents viral protein maturation
How do viruses reproduce
- viral genome enters host cell
- replicated to produce multiple copies –> transcribe and translated to produce viral coat protein
- viral genomes assemble spontaneously with coat protein to create virus particles
- particles escape cell via lysing (some DNA viruses remain latent for sometime)
Why is it that closely related organisms have similar DNA sequences and genomes
Conservation of genetic information
- some DNA sequences that code for important cell functions and behaviours are conserved (ex. small ribosomal subunit RNA)
- exons are conserved the longest (across all species)
- intron conservation is also observed in mammals (species that are closely related to eachother have similar intron sequences)
Vital statistics for human genome
- 21 000 protien coding genes
- 1.5% DNA sequence is exons
- 3.5% conserved with other mammals (including regulatory genes)
- single nucleotide alterations occur on average about 1 / 1000 nucleotides
Explain steps involved in Sanger Sequencing and how to read a sequence off a gel
1) Primer
add primer to single stranded DNA
(primer sequence often determined by the restrictions where DNA is cut)
2) Chain Termination
dideoxy-nucleotides (no 3’ hydroxyl group) block DNA polymerase once incorporated
(DNA fragments are at diff lengths and ends with a specfic 3’ terminal dideoxy-nucleotides)
3) Electrophoresis
electrophoresis used to separate the differently sized DNA fragments. Fragments move towards positive end of electrophoretic gel + shorter fragments move fastest
Explain Steps involved in Second/ Next Generation Sequencing
(how is diff from Sanger?)
(what is the importance of sequencing overlapping genomic fragments?)
1) Start
thousands of overlapping genomic fragments are placed on a spot on a micro-fabricated slide
- initiate reaction with primers (complementary sequence added in cloning step)
2) Stop + Pose
FLUOURESCENT reversible chain terminator dideoxy-nucleotides (dNTPs) are added
- can be digitally photographed once added to chain by DNA polymerase
3) Go again
fluorescent tags removed ( the nucleotide no longer terminates teh chain due to chemical modification)
- another fluroscent termination nucleotide is added to 3’ based on sequence
importance of overlapping sequenced gragments
- if cut in parallel –> requires lots of computing power)
- overlapping fragments can allow for more accurate and increased reads of each fragment –> greater depth)
Identify what information about DNA is measured in Third Generation Sequencing.
How does this method differ from other strategies?
Examines intact DNA fragments directly
- DNA fragment is pulled through a nanopore and causes chanes in optial properties / ion fluxes
- reads both DNA sequence + nucleotide modification that alters transcription of coding regions
- DNA passes through nanopore
- Raw ouput: electrical signal caused by nucleotide blocking ion flow
- each nucleotide has specific electric “signature”
(differs from other strategies because it doesn’t require DNA synthesis)
Define ‘restriction (endo)nuclease’ and describe the relationship between the type, and frequency of ‘cuts’ to each enzyme based on the number of nucleotides recognized)
restriction endonuclease:
- enzymes that restrict transfer of DNA from one species of bacteria to another via cutting DNA into pieces
- used in genetic engineering:
- recognize specific internal DNA sequences (4 - 8 nucleotide base pairs)
- make reproducible cuts at the same restriction sites –> isolation of smaller DNA sequences used in DNA cloning
Different endonuclease:
(frequency)
- four base pair restriction nuclease cuts 1 / 256 nucleotides
- sic base pair restriction nuclease cuts 1 / 4096 nucleotides
(type)
- blunt ends
- 5’ overhangs –> used to facilitate joining of 2 diff pieces of DNA cut with same nuclease with high fidelity
Define Cloning and Recombination
Cloning:
production of identical copies of DNA (amplification)
recombination:
cloned DNA is amplified in bacteria after being shuttle into bacterial plasmids
describe steps in cloning a gene fragment from bacterial plasmids
Plamids consist of few thousand base pairs of circular DNA
linearized by restriction nuclease digestion + recombined with DNA sequence fragment to be cloned
- Cut
plasmid and DNA fragment to be cloned cut with the same restriction nuclease to have stick overhangs (used to base pair with eachother) - Paste
After basepairing, plasmid DNA fragment is ligated together from 5’ to 3’ on each strand –> results in recombinant DNA - Amplified
recombined plasmid DNA amplified in bacterial cultures:
recombinant DNA is introduced into bacteria cell –> undergo cell division to clone the DNA of interest
Compare + contrast ‘genomic’ and ‘cDNA’ libraries
genomic libraries:
- genomic DNA cut with restriction nuclease + all fragments recombined into plasmids (which are amplified in bacteria)
- specific DNA clones in library containing sequences are identified via hybridization probes
complementary cDNA libraries
- cDNA sequenced by RNA sequences
(changes to cells’ mRNA population due to development, physiological / disease can be assessed by generating cDNA libraries)
- complementary cDNA strand generated using reverse transcriptase (RNA –> DNA)
- og mRNA degraded via RNAse + second strand of cDNA generated using DNA polymerase
Both:
- cDNA’s can be cut with restriciton nucleases and made into libraries
define hybridization probe + possible sources
DNA probe:
- single stranded DNA
- double stranded DNA is denatured (heat or acidity) to generate single strand DNA
- single strands when cooled + or at natural pH state will come together (renature) on basis of sequence-specific nucleotide base pairing
- if denatured single DNA strands are fixed onto surface : labelled complenetary single strand DNA probe used to identify specific DNA sequence / cone amongst large mixture of DNA sequences or clones via renaturation
possible sources:
- other genomes:
- RNA that has been reverse transcribed into DNA
- amino acid sequences in proteins
- databases
explain steps involved in probing library to identify target gene sequences
- place absorbent paper on top of a petrid dish with colonies of bacteria containing recombinant plasmids (library)
- peel paper off from dish –> produces replica of colonies
- bacteria lysed and DNA denature with alkali (DNA is bound to paper)
- radioactively labeled DNA probe is added, incubated with colonies on paper and washed
- Paper is exposed to photographic film (positions of desired colonies detected by autoradiography)
explain how probes can be used ‘in situ’ to identify genes and transcripts in cells and tissues
labelled probes:
1. used to determine localization of DNA / RNA within tissues / cells (ex. identify virus-infected cells)
- used to determine localization on chromosomes in cells / tissue
- differently colored probes attach to genes on maternal and paternal copy of chromosome of interst in nromal cell (visualized at methaphase of mitosis after DNA replication) - used to identify chromosomal translocations in cells / tissues
Explain sequential steps in each PCR cycle of amplifcation
Polymerase Chain Reaction: used to generate genomic or cDNA clones from complex mixture of nucleic acid fragments
Step 1.
add heat to separate double strand into single strands
step 2.
cool strands to anneal primers on each strand (cools to temp such that primers anneal first before the complementary strands)
step 3.
use DNA polymerase to initate DNA synthesis
step 1.
add heat to separate double strands…
1st cycle:
- amplification of two new DNA strands of DNA sequence are cloned (generates extended ends downstream of primers)
- intially, the clones are longer than the target sequence but each cycle slowly clones the right size
Each cycle produces an exponential amount of DNA clones:
After 3 cycles: the single DNA strands cloned are identical to the target sequence
Define expression vector, recombinant protein and chimeric reporter genes
Expression vector:
- used to produce recomibnant proteins in bacteria, yeast, insect or mammalian cells
- specially designed vectors that induce transcription + translation signals to guide inserted gene that is meant to be expressed at high levels
recombinant protein:
- proteins expressed by recombinant DNA
chimeric reporter gene:
- gene encoding a protein whose activity is easy to monitor experimentally –> used to study the expression pattern of a target gene / localization of protein product
