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What is the role of DNA ligase in vivo?
It will synthesize missing phosphodiester bonds in a DNA strand
What is a use of DNA ligase in vitro
It can bond two DNA strands together
Why are sticky ends more ideal than blunt cuts?
Sticky ends ensure the DNA is being inserted in the right orientation. With blunt ends there is the chance that the DNA strand will flip
DNA polymerase
An enzyme that synthesizes DNA
template-dependent DNA polymerase
An enzyme that copies an existing DNA or RNA molecule
What are the two features of template-dependent DNA polymerase?
Unable to utilize an entirely single stranded molecule as a template (aka it needs a primer!)
They can synthesize and degrade nucleotides. During proofreading in the 3’ to 5’ direction, if a base needs to be removed due to a mistake in synthesis, they need exonuclease activity (5’ to 3’ exonuclease activity is rare)
What are the two roles of primers?
Delineates the DNA
Provides a space for the polymerase to attach and begin synthesis
How do you make primers for unknown genomes?
Attach linkers to area where restriction enzymes cut, so you will know the sequence
During the annealing phase, how do you ensure that the primers anneal and not the DNA strands rejoining?
Short primers are faster at finding their spot and annealing
Why does exonuclease activity tend to not be in the 5’ to 3’ direction?
This may degrade the 5’ end of a polynucleotide that has just been synthesized
All enzymes are proteins except for what?
Ribozymes, which is RNA
What is a clone library?
a collection of clones whose inserts cover an entire genome
What is a cDNA library?
A library of just genes, the introns have been cut out
How does a cDNA library differ from a genomic library?
cDNA is just genes, while the total genomic library contains all genetic material including introns and exons
What is a Southern Hybridization aka Southern Blot?
A technique used to identify a segment of DNA or gene of interest
How does a Southern Blot work?
After we have run a gel we will
place a gel on a support with the nylon membrane on top
Place paper towel and then a weight on top of the membrane
moisture gets sucked up though the wick, the gel, then the membrane taking 1. the DNA from the gel
Probes are added to the membrane
Extra probes get washed off membrane
Probes will stick to our desired gene and then with autoradiography, the probes will show up on xray film
Why do we map genomes?
It enables us to identify unique features of that genome and orient us to the genome
^
It provides a guide for sequencing experiments
What is the standard method for sequencing prokaryotes?
Shotgun sequencing
How does shotgun sequencing work?
Random DNA fragments are created with sequence overlap between the fragments, so that the fragments can be put back together to look at the genome
What are the disadvantages to shotgun sequencing?
Errors are high when repetitive sequences of the genome are analyzed
The data analyses become disproportionately complex with larger genomes
What is a sonicator’s role in shotgun sequencing?
The sonicator will use sound to basically agitate the DNA so much that it breaks the DNA apart
What does Botanga mean when he says that data analyses for shotgun sequencing can become disproportionately complex
The complexity does not follow a pattern, so data analysis for a larger genome can get extremely complicated really quickly
What are tandem repeats?
When a pattern of two or more nucleotides is repeated and the repetitions are directly adjacent to each other
What is a VNTR? and what is its other name?
Variable number tandem repeat also known as a minisatellite is a tandem repeat that varies between 14 and 25 nucleotides repeated
What is an STR? And what is its other name?
Short tandem repeat also known as a microsatellite has repeats with fewer than 14 nucleotides
What are the two forms of sequencing for eukaryotes?
Whole genome shotgun method
clone contig method
important factors for the whole genome shotgun method
Involves random sequencing of entire genome
Most applicable where there is an existing reference sequence
Markers are used as landmarks to anchor the assembled sequences on to the map
important factors for the clone contig method
Genome is broken up into manageable segments
starts with segment of DNA whose position on the map has been identified
This step-by-step process is slower, but more accurate
What kind of genes are useful as markers?
The gene must exist in two forms or alleles, each specifying a different phenotype
What does one do if phenotypes are too complex, difficult to distinguish, or too few visual phenotypes for a gene?
Use biochemical markers
What are the two methods of mapping?
Genetic mapping and physical mapping
What is genetic mapping?
It uses genetic techniques to construct maps showing the position of genes and other sequences on the genome
What is physical mapping?
It uses molecular biology techniques to examine DNA molecules and construct maps showing the positions of sequence features, including genes
What are the shortcomings of using genes as markers?
They do not show the intergenic region, which makes up a large chunk of the genome
Few genes have the nice allele system with distinct phenotypes
What is the difference between a character and a trait?
A character is a phenotype being observed like height, weight, eye color. Traits are the options for those characters, so tall/short, fat/skinny, blue/brown
How do we use genes as markers?
We make crosses, look at progeny, and see how they are segregating (segregation pattern), and look at the genetic distance between different genes
What are the four types of DNA sequence features that are used for physical mapping markers?
Restriction fragment length polymorphisms (RFLPs)
Simple sequence length polymorphisms (SSLPs)
Single nucleotide polymorphisms (SNPs)
Amplified fragment length polymorphisms (AFLPs)
What is AFLP and how does it differ from RFLP
This is a PCR based approach (unlike RFLP) that uses rare and frequent cutters to detect polymorphisms in a genome with no information
Trace amounts of DNA will still work with this method since it uses PCR
What are the two methods of scoring an RFLP?
Southern blot using a probe
Using PCR primers that are designed to anneal on either side of the polymorphic site
What is scoring?
Similar to typing, it is how we analyze and interpret the pattern created for example by an RFLP
What is RFLP?
RFLP are polymorphisms where different alleles may have restriction enzyme cutting sites either present or absent
Used for forensics
What is an SSLP?
These are arrays of repeat sequences that display length variations
Why are STRs more popular than VNTRs for SSLPs?
VNTRs are not evenly spread in the genome (mostly in the telomeric region), unlike STRs
STRs are shorter and therefore a lot faster and more accurate
What are the two ways to type SSLPs?
Agarose gel electrophoresis
Capillary electrophoresis
Where are primers designed for SSLP?
They are designed just outside of the repeat region
What is typing?
Typing is used to screen different genes in a population
What is capillary electrophoresis
DNA in buffer uses capillary action to go through small tube with a detector in it, DNA lengths can then be seen with computer software
What is a SNP?
Single nucleotide differences among individuals
What is the frequency of SNPs a eukaryotic genome?
about 1 for every 10kb
What is an oligonucleotide?
A short single-stranded DNA molecule usually less than 50 nucleotides in length
Why is oligonucleotide hybridization high stringent?
Hybridization will not occur unless the oligonucleotide is able to form a completely base-paired structure with the target DNA
What is solution hybridization and what is it used for?
Solution hybridization is a method for finding SNPs. An oligo with a florescent dye and a quenching compound is mixed with DNA and when the oligo lines up with the SNP segment, it will separate the dye and quenching compound and start fluorescing
How to DNA Microarrays/DNA Chips work?
They allow many hybridization experiments to be performed in parallel
A variety of different probes are on the chip and when labelled DNA is pipetted onto the chip SNPs will be immobilized at defined positions on the chip
This also can be used to observe changing levels of expression in candidate genes
What is a hybridization with an oligonucleotide with a terminal mismatch?
When there is a SNP at the end of an oligonucleotide, the oligonucleotide will hybridize and have a non-base-paired tail
What are the two tests that utilize oligonucleotide terminal mismatches
oligonucleotide ligation assay
ARMS (Amplification refractory mutation system) test
How does an oligonucleotide ligation assay work?
Use two oligos that anneal at the site of a SNP, if the SNP is there, ligation will occur making a one long oligo, if the SNP is not there then ligation cannot occur so we end up with two shorter oligos
This is typed using capillary electrophoresis
How does an ARMs test work?
primer for PCR is designed to anneal to SNP. If SNP is not there then primers cannot anneal we get no PCR product for that strand
Iinkage Analyses
The basis of genetic mapping
What are pure-breeding types?
Homozygous individuals
What is crossing over and when does it happen in meiosis?
Crossing over is the exchange of genetic material between homologous chromosomes, this occurs in prophase I of meiosis
What are the different linkage analyses and which organisms are they ideal with?
Breeding experiments ← mice, flies
Pedigrees ← Humans
Biochemical markers with conjugation, transduction, transformation ← bacteria
How does recombination frequency help with mapping?
The frequency with which the genes are unlinked by crossovers is proportional to how far apart they are on the chromosome
What are recombination hotspots?
Regions are chromosomes that are more likely to be involved in crossing over
What is a Pedigree?
a family tree that describes the genetic interrelationships of parents and children across generations
What is it called when pedigrees are missing generations and how can you use them for analyses?
Imperfect pedigrees, you can analyze them statistically by using the lod score
what does lod score stand for?
logarithm of the odds
What are the 3 ways by which bacteria transfer DNA?
Conjugation, transduction, transformation
What markers are used to map bacterial genomes?
Biochemical markers
What is conjugation?
The transfer of DNA between two bacteria using a sex pillar (closest to what we would think of as sexual reproduction)
what is transduction?
The transfer of DNA from one bacterium to another via a bacteriophage
What is transformation?
A situation let’s say where bacteria dies, and its DNA is left in the environment and then picked up by another bacteria
Why do you need a physical map to do with a genetic map?
Resolution of the genetic map depends on the number of crossing over scored
Genetic maps have limited accuracy
What are the three common physical mapping approaches?
Restriction Mapping
Fluorescent in situ hybridization (FISH)
sequence tagged site (STS) mapping
How does FISH work?
Uses fluorescent probes to help detect and localize presence or absence of specific DNA sequences on chromosomes
How does STS work?
It is a short DNA sequence that has a single occurrence in the genome and whose location and base sequence are known. These are detected using PCR
How does chain termination work?
Start will multiple identical, single-stranded DNA molecules
Anneal short oligos to the same position on each molecule
DNA polymerase synthesizes DNA with dNTPS until it uses a ddNTP then the synthesis terminates
Each ddNTP is labelled with different fluorescent markers and fragments with those markers will be picked up in a polyacrylamide gel
What happens if during chain termination, the software is not confident what the base is?
It will have low squiggles and be labelled N
What structure in ddNTPS cause the termination of synthesis?
There is a H instead of OH group in the c3 prime region
If synthesis during chain termination is terminated when a ddATP is incorporated, then what was the base on the template DNA strand?
dTTP
What are the chances of the DNA polymerase picking a dNTP versus a ddNTP?
50/50 the DNA polymerase does not discriminate between the two
How do you obtain single stranded DNA from m13/e coli?
DNA is inserted into M13 bacteriophage genome which then infects ecoli cell
Ecoli replicates this plasmid, which leads to some double stranded DNA and some single stranded
New phages are released with copies of single stranded DNA in its protein coat
What is the disadvantage of using ecoli/bacteriophages to create single-stranded DNA?
DNA fragments longer than 3kb suffer deletions and rearrangements
What is an alternative approach to obtaining single-stranded DNA?
Single stranded DNA can also be obtained via denaturation with alkali
What is a universal primer?
a primer that anneals to vector DNA right before the DNA insert. It is called universal because you can put any kind of DNA in the insert region and it will be synthesized
Why do you need internal primers?
Polymerase can only go so far (1000bp) before it falls off, so internal primers can help keep synthesis of DNA insert going
How do you design an internal primer?
If the polymerase stops at 1000 bp into your insert, design your internal primer to start at 950bp, if that stops at 1950bp, design the next internal primer to start at 1900bp, repeat the cycle until the end of your DNA insert
What are the three needs for an enzyme when conducting sequencing?
High processivity
negligable or zero 5’ → 3’ exonuclease activity
negligible or zero 3’ → 5’ exonuclease activity
Why do you want high processivity in a sequencing enzyme?
Because that means the enzyme can go many nucleotides before falling off
Why do you want little to no exonuclease activity in sequencing enzymes?
You do not want proofreading to mess up the template nucleotide sequence or fix a mutation that is there
test cross
A cross that allows us to determine the genotype of an individual showing a dominant trait (homozygous or heterozygous)
Qualitative versus quantitative genes
qualitative has one gene for one phenotype for one gene
quantitative has many genes for one phenotype, which leads a slew of phenotypes
