Molecular Techniques Flashcards
(100 cards)
1
Q
What is cloning?
A
taking a gene from its native source and moving it to a new vector (such as plasmid).
2
Q
Where do restriction enzymes originate?
A
Part of the restriction/modification system of prokaryotic organisms
3
Q
What type of restriction enzyme is most often used?
A
That which produces sticky ends and has high specificity to one place in the DNA
4
Q
Describe restriction enzyme/ligation cloning.
A
- cleave DNA of interest with restriction enzyme
- Cleave cloning vector with the same restriction enzyme
- remove cut part of plasmid with electrophoresis
- recreate closed, circular dsDNA by linking the cut parts of DNA using ligase (ATP-dependent)
- transform bacteria and use positive selection encoded in plasmid to select for ligated molecules
5
Q
Describe cloning vector pUC57
A
- oriC
- amp resistance
- cloning region with many restriction sites
- B-galactosidase for color screening (white colonies are cloned, blue are not)
6
Q
What are the natural processes by which foreign DNA gets into bacteria cells?
A
Transformation = DNA in surrounding environment
Conjugation = sex pillus
Transduction = bacteriophage
7
Q
How can transformation be promoted in E. coli?
A
It is not normally competent, so we must pre-treat with calcium or electroporation.
8
Q
What are two common bacteria cloning strains?
A
- DH5a
- TOP10
these are mutated to lessen homologous recombination and endonuclease activity.
9
Q
What is a common protein expression bacteria strain?
A
BL21(DE3):
- lacking proteases
- optimizes T7 polymerase/lac operon systems
- can incorporate chaperones, disulfide isomerases, rare tRNAs
10
Q
Describe the E. coli expression vector.
A
- oriC
- promotor
- affinity tags
- coding sequence for tag removal
- multi cloning sequence (restriction sites)
- inserted gene sequence
- selection marker (can be cut out later)
11
Q
What is the purpose of making a genome library?
A
To find a gene sequencing
12
Q
Why are genome libraries not often used anymore?
A
Because we do DNA sequencing and PCR more often
13
Q
Describe how to make a genome library.
A
- randomly shear DNA with 4-bp cutters for a partial digest (will cut once every 256 base pairs to generate DNA fragments in uniform length)
- fragments are then inserted into vectors
14
Q
Describe the vector types used to make a genome library.
A
- plasmid: limited to 10,000 bp of DNA
- phage: can package larger pieces of DNA into phage (30,000bp)
- BAC: bacterial artificial chromosome, can hold 100,000bp. hard to purify because it is present in only one copy in the bacteria
- YAC: yeast artificial chromosome, can hold several million bp.
15
Q
Why do we make cDNA?
A
It can give us a look into what RNA is expressed at a certain time by making a more stable nucleic acid
16
Q
How is cDNA made and cloned?
A
- oligoT primer hybridizes to the poly A tail of mRNA
- reverse transcriptase synthesizes DNA, using mRNA as a template, in 5’->3’ direction
- RNA is degraded with RNAseH, leaving behind a small RNA primer to use in synthesis of second DNA strand
- second strand of DNA is synthesized by DNA polymerase
17
Q
Describe the PCR reaction.
A
- heat to separate strands (denaturation)
- cool to anneal primer pair (annealing)
- synthesize DNA with temperature stable polymerase (elongation)
18
Q
How many reps of PCR are performed?
A
About 30. Each replicate doubles what we have (2^n)
19
Q
Describe the drawbacks of Taq polymerase
A
- error prone and lacks 3’->5’ proofreading activity
- can only do short products
we therefore will compensate with other polymerases
20
Q
What are the benefits of using vent polymerase?
A
has 3’->5’ proofreading exonuclease
-has high thermostability: long half-life
21
Q
What is the benefit of using long-range polymerase?
A
-can amplify up to 30k (solution to short products by Taq)
-
22
Q
How does one select for a good primer pair?
A
- melting temp between 50-65 (two primers need same Tm, and we reanneal at 4 degrees below this)
- absence of dimerization capability between the pair
- absence of hairpin formation within a single primer
- lack of secondary priming sites
- primer length (18-22bp)
23
Q
How can PCR be used to diagnose duchennes muscular distrophy?
A
you can amplify exon 46. In those with the disease, when you amplify with PCR and run on a gel there will be no band when you probe for the exon.
24
Q
How can PCR be used to add sequences?
A
- Because the 3’ end of the primer needs to be stable but the 5’ end does not, we can get an overhang at the ends of the DNA sequence we want to add to.
- the overhang will then be filled in by DNA polymerase, and the new ends are made (perhaps with new restriction sites or a tag)
25
When would we use PCR to add sequences?
If we want to clone a piece of DNA into a vector but it does not have the restriction sites we need to do so.
26
How can we tell if a yeast gene is successfully knocked out using PCR?
We can use a positive selection marker to replace the gene being deleted. PCR can be used after digestion to look for difference in restriction digest bands.
27
What is a positive yeast selectable marker?
kanomycin
28
What is another name for direct PCR cloning?
Ligation independent cloning
29
Describe ligation free cloning
- cut cloning vector with restriction enzyme
- prepare insert with several base homology (~15bp) to the vector ends using PCR.
- mix insert with linearized vector
- treat with T4 DNA polymerase to chew back dsDNA (3'-5' exonuclease activity) and leave 3' overhangs that anneal together
- then put in E. coli to ligate, because it has its own ligase
30
What are the cons to ligase-free cloning?
efficiency of cloning is only 10-25%
31
What are the benefits to ligase-free cloning?
- high throughput, so can screen for many transformed colonies at once using PCR
- can quickly and cheaply clone same insert into many types of vectors and is cheap!
- can clone many insert boundaries into the same vector which is good for screening functional regions of a gene
32
How is In-Fusion enzyme used?
it works like T4 DNA polymerase to do ligase-free cloning of multiple gene fragments all in one tube
33
What is assembly PCR good for?
Production of synthetic genes and even synthetic genomes.
34
How does assembly PCR work?
- oligonucleotides with overlapping regions are extended in PCR
- primers at the end of the DNA molecule allow for amplification of the target sequence
35
What is codon optimization?
switching the codons used in a transgene without changing the amino acid sequence that it encodes for in order to replace rare codons with those matching more common tRNAs. Increases the abundance of the protein expression dramatically
36
What is allele-specific PCR?
It is a diagnostic or cloning technique based on single nucleotide variations. A specific primer can only amplify a normal or mutant allele, but not both within the same reaction.
37
What is nested PCR used for?
It reduces non-specific binding in PCR products due to the amplification of unesxpected primer binding sites.
38
How does nested PCR work?
- Two sets of primer pairs are utilized
- the first round of PCR may have nonspecific primer binding elsewhere than the target sequence resulting in an unwanted product
- second round uses different primers that won't hybridize to the first unwanted product, but will hybridize within, or nested in, the first, wanted product.
39
What is degenerate PCR?
Uses degenerate PCR with primers that are degenerate in their 3rd base to amplify DNA sequences that are unknown but related to a known sequence. Degenerate primers are a mix of primers that differ in the last position.
40
What is RT-PCR useful for?
- detecting transcripts of really any gene
- eliminates need for abundant starting material for northern blot
- provides tolerance for RNA degradation as long as RNA sequence spanning the primer is in tact.
41
What are the two starting materials for RT-PCR?
- mRNA
- cDNA-\>mRNA
42
How is gene expression measured using RT-qPCR?
- Incorporate sybrgreen into dsDNA as it is replicated. More fluorescence from sybr green indicates more DNA. the earlier the fluorescent midpoint (i.e. less cycles), the more DNA there is.
- OR, Taqman probe bound to DNA being amplified. as it is amplified, the probe is displaced and cleaved, causing it to emit fluorescence. Measured and interpreted the same way as sybrgreen
43
In addition to measuring the expression of mRNA using qPCR, what else do you measure?
expression of a housekeeping gene such as GAPDH as a control
44
Describe Sanger DNA sequencing.
- anneal primer to DNA sequence
- DNA polymerase extends the primer using regular nucleotides and ddNTPs that are 32P labelled (not many of these), and uses these at random
- the ddNTPs when incorporated terminate the elongation.
- then run gel. Each lane is a different reaction using A,T,C or G. The furthest band to run is the earliest in the sequence. (end of gel = 5', beginning of gel = 3')
45
Which ddNTP is 32P labelled for Sanger sequencing?
the alpha phosphate (the one left behind after cleavage)
46
Describe automated dideoxy sequencing.
- done like PCR, but only one primer set so that amplification is not exponential
- uses ddNTPS with each a different color fluorophore so the reaction can happen all in one tube.
47
How is DNA sequencing performed from DNA libraries?
- The genome is already fragmented in vectors in the library
- overlapping fragments that are contiguous are called contigs
- contigs that are nearby but have small gaps are called scaffolds
48
what is sequencing coverage?
The number of times each base is sequenced
49
In DNA sequencing, what is a read?
It is the sequence of a single DNA fragment. These will form contigs. Contigs will form scaffolds.
50
What is a difficulty of sequencing from a DNA library?
DNA repeats are hard to order in the genome, as well as heterochromatic regions, so these oftentimes are absent from the final genome sequence.
51
What is an EST?
Expressed sequence tag from mRNA, and is specific to the cell.
52
What is a tilling array?
It is a whole genome microarray, but the probe design is different. The tilling probes are from known contiguous sequences. Resolution power depends on probe design, i.e. how much they are spaced apart or are overlapping.
53
What is a tilling array used for?
It is used to determine which parts of a sequence are coding. We probe to cDNA, i.e. ESTs (expressed sequence tag from mRNA)
54
What types of things do you look for in a new genome?
- microRNAs
- protein coding sequences
rDNA loci
tRNA genes
-transposable elements
55
What are the attributes of a compact genome?
- small gene size
- small exon size
- small intron size
- small intergenic regions
56
Describe the daphnia genome in one word.
compact
57
Describe, in general, what next generation sequencing technologies do.
Generate millions to billions of small sequencing reads that are alligned against a reference sequence (one that is similar, or in the case of sequencing cDNA, the known genome sequence)
58
What is the basis of de novo sequencing?
Overlapping of reads.
59
What does 'paired ends' mean?
The two ends of the same DNA molecule. YOu can sequence one end, then turn the molecule around and sequence the other end. The two sequences you get are "paired end reads"
60
What is the general outline of high throughput sequencing?
- obtain DNA or RNA (requires conversion to cDNA) from single cell or population of cells
- PCR
- DNA is fragmented and ligated to adaptors specific for to the sequencing procedure. (adaptors are needed for clustering)
- sequencing is performed using "sequence by synthesis" (a variation of Sanger sequencing)
61
Describe clonal single molecule array
Uses adaptors to fill a chip with amplified DNA strands
62
Describe Illumina's sequencing reaction.
Sequencing by reversible termination:
- one round of synthesis, termination at adaptor, capture of fluorescence
- do the same with the reverse strand
- you have built up a series of base calls that are position-specific based on the size of the read, and align them to a reference sequence
63
What is a drawback to illumina sequencing?
Reads are short, at only 50-250bp.
64
What are the fast and slow parts of illumina sequencing?
Fast: the actual sequencing
slow: the data analysis
65
Describe single molecule real-time sequencing (SMRT sequencing)
Based on replication of DNA in living cells. Watches which nucleotides DNA polymerase incorporates in real time. Each nucleotide in the nucleus is given a different fluorescent tag. Fluorescence on terminal phosphate, so when it is cleaved away, fluorescence is emitted and measured. Zero mode waveguide measures this. Can use thousands of these ZMWs at a time.
66
What are the benefits of SMRT sequencing?
High speed, long read length (high quality de novo genomes, span repetivie elements and complex regions, unambiguously align sequences), high fidelity. Overcomes bias in AT or GC rich regions.
The cost is also currently decreasing
67
How can we study relationships between different malaria parasites?
Phylogenetics
68
What is the human metagenome?
Homo sapiens genes and genes present in the genomes of trillions of microbes that colonize our adult bodies
69
In general, how is the human microbiome sequenced?
-amplification and sequencing of 16S rRNA to tell us what species the DNA came from, or can do total genome sequencing, both using degenerate primers
70
How was it determined that certain bacteria are capable of expressing enzymes that metabolize cancer chemotherapeutic drugs?
By sequencing 16S rRNA of microbiome using illumina sequencing. Whole genome sequencing corroborated this, showing that sequencing of 16S rRNA is sufficient.
71
Why might we want to do exome sequencing rather than whole genome sequencing in humans?
- the majority of human DNA are not genes, but repetitive regions
- studies of mRNA are compromised by expression being tissue specific and time specific, and exome sequencing allows us to determine what the genes are without studying mRNA
72
What are the two methods of exome sequencing?
1. amplicon-based
2. capture hybridizatin-based
73
General steps of exome sequencing?
- select subset of DNA that codes for proteins
- sequence exonic DNA using high-throughput DNA sequencing technology
74
Descrbe amplicon-based exome sequencing.
1. digest cDNA (only protein coding DNA!) with restriction enyme and denature
2. hybridize probe library (probes are biotinylated for purification)
3. amplify with PCR
4. high-throughput sequencing
75
What is a barcode sequence?
A Barcode sequence is a short nucleotide sequence from a standard genetic locus for use in species identification.
76
What types of proteins are we using ChIP-seq for mostly?
transcription factors
77
How does ChIP Seq work?
Combines chromatin immunoprecipitation with massively parallel DNA sequencing to identify binding sites of DNA-associated proteins.
- cross link and fractionate chromatin bound to the protein to maintain the interaction
- immunoprecipitate protein with antibody and reverse cross link so we are left with just the DNA
- end repair the DNA and phosphorylate the ends
- add primer linkers for amplification and sequencing
- HTS and comparison to control to make sure there are no non-specific interactions
78
What are three ways to measure gene expression?
- northern blot
- qRT-PCR
- gene promotor fusions to reporters (luciferase, GFP)
79
How do we use microarray analysis to study whole genome expression?
- DNA oligos are printed onto high density array chips
- gene expression can be measured now under varying conditions (e.g. developmental stages, pathogenic vs normal states, cancer genomics)
80
What are the different types of arrays?
- glass arrays (spotted on glass microscope slides); spot oligonucleotides derived from the genome, or PCR products from cloned DNA library in the old days)
- DNA chips (oligos are directly synthesized onto the chip)
81
How do we compare gene expression of a normal vs pathogenic state?
take cDNA and probe it in the region of interest with different colored probes. then hybridize to the microarray chip and look at difference in color (red vs. green vs. yellow)
82
How are oligonucleotide probes designed for microarray?
1. Need 70bp probes. Start by looking at all 70mers in the coding region
2. Look for uniqueness usuing Blastn
3. Look for patterns to avoid (high GC regions)
4. avoid self-binding
5. make sure it is complex enough
6. make sure all probes have same Tm (i.e. similar GC content)
7. average all of these scores and choose the best ones
7. make sure cDNA hybridizes best to 3' end
83
How is a microarray beneficial over northern blot?
You can look at several thousand genes at a time. We just can't quantify it
84
How is total RNA extracted?
TRIZOL extraction
85
What is the benefit of using RNA-seq over microarray analysis?
You get the same results, but RNA-seq is cheaper and more sensitive
86
What type of sequencing does RNA-seq use?
It can use any, like illumina, because it has been converted to cDNA
87
Describe the Affymetrix DNA chip
Oligos 25bp or shorter are synthesized directly on the chip by masking certain spots and adding certain nucleotides at a time
88
What is the advantage of the affymetrix DNA chip over a glass slide?
You can fit a much higher number of oligos on the chip and can therefore use multiple probes for the same gene
89
What is a downside to the Affy chip?
There is only one color probe, so you need a different chip for every condition (i.e. for a 48 hour experiment, need 49 chips including the control)
90
How does the affy chip and oligo glass slide differ in terms of probes?
Number of colors, length, how they are synthesized
91
How can a high density array be used to direct allelic variation in the yeast genome?
- use microarray chips that can resolve single base pair change
- probes were 25-mers to increase sensitivity (compared to 70mers)
- probes were used as genetic marker between yeast genomes
Can also use this to determine inheritance patterns of yeast chromosomes (mom or dad?)
92
How is a chromosomal microarray (CMA) used in humans?
It is used to see chromosomal sequence and copy variations
93
What may individual responses to drugs be attributed to?
SNPs in the human genome
94
What is the average difference in human genomes?
1 SNP in every 200-300bp
95
How can human SNPs be detected?
Affy chip!
96
How can SNPs locate genes in DNA sequences?
e.g. identify SNPs in people that have high blood pressure vs those with normal blood pressure. A more common SNP can point to the gene and its location using affy chip (look for location of probe to which the DNA that has the SNP is bound)
97
How are SNPs detected using high resolution melting?
In general, we are looking at differential melting of DNA
- ampliy small fragments of DNA containing SNPs in the presence of SYBR green (binds to dsDNA)
- denature and rapidly cool, allowing for the potential of a mismatch
- slowly increase heat and monitor SYBR green fluorescence
- where there is a mismatch, the fluorescence will decrease
- overall, relies on the fact the SYBR green only intercolates into the DNA when there is correct base pairing
98
What is a drawback of the affy chip?
The chip and the detector are both expensive
99
What is the advantage to using using high resolution melting to identify SNPs compared to microarray?
It is cheaper and faster, and does not require sequencing
100
What is a drawback to using microarray for transcriptome sequencing?
It relies on what is already sequenced to make the probes. So, if the sequence of a region is not known, you may miss identifying part of the genome that contributes to the transcriptome.
