CReATe Research - Genetic Testing

omarys's version from 2017-08-04 17:48

Section 1: Karyotyping

Question Answer
allows for visualization all of the human chromosomes at one time by "painting" each pair of chromosomes in a different fluorescent colorSKY (spectral karyotyping)
allows for viewing the full set of human chromosomes in black and white, a technique that is useful for observing the number, size and shape of the chromosomestraditional karyotyping
Can chromosomal translocations be identified by SKY?Yes, by visualizing a chromosome (painted in one color) with a small piece of a different chromosome (another color) attached to it.
Disadvantage: The conventional karyotype could be normal if the deletion/duplication in the patient with pathological phenotype (patient with mental retardation, autism, epilepsy, etc.) is ___ than its ___ (about ___).lower ; resolution ; 10Mb

Section 2: FISH

Question Answer
FISH is a ___ techniquecytogenetic
Binding ___ are used to determine ___ ___.probes ; sequence complementarity
To find out where the fluorescent probe bound to the chromosome, ___ ___ is used.fluorescence microscopy
FISH is an important tool for understanding a variety of ___ ___ and other genetic ___.chromosomal abnormalities ; mutations
FISH is different from most other chromosome study techniques in that it has no need to be performed on cells that are ___ ___, making it a more ___ procedure.actively dividing ; versatile
Summary scheme
An example of when FISH is useful is identifying ___ a particular gene ___ within an individual's ___.where ; falls ; chromosomes
In FISH, a probe that is ___ to a ___ sequence is made, and then labeled with a fluorescent marker.complementary ; known
3 types of FISH probes w/ different applications exist: ___-specific probes, ___ or centromeric repeat probes, and ___ ___ ; alphoid ; whole chromosome
useful when researchers have isolated a small portion of a gene and want to determine on which chromosome the gene is locatedlocus specific probe
used to determine whether an individual has the correct number of chromosomes, or in combination with locus specific probes to determine whether there is missing genetic material from a particular chromosome.centromeric repeat probe
useful for examining chromosomal abnormalities, e.g. translocations; their use yields a full-color map of the chromosome, i.e. SKYwhole chromosome probes (they are collections of smaller probes)
The FISH technique is another methodology that allows for detecting ___ in ___ genomic regionsrearrangements ; specific
FISH is generally used for “___ investigations” based on a ___ clinical ___.targeted ; specific ; indication (This limits the application of FISH, as in many cases pathological phenotypes are not related to known syndromes.)

Section 3: CGH

Question Answer
aCGH is the FIRST genetic test for the diagnosis of genetic syndromes with ___ ___, congenital ___, and ___ disorders.mental retardation ; malformations ; neurological
The array-based CGH is AKAmolecular karyotype
identifies DNA anomalies known as ___s, which are not detectable by other conventional cytogenetic techniquesCNV
CNVs are due to the ___ of genomic portions (___) or to the presence of ___ ___ of DNA segments (___).loss ; (deletions) ; extra copies ; (duplications)
aCGH analysis guarantees a much ___ ___ compared with other genetic investigations, e.g. karyotype analysis and FISH.higher resolution
aCGH detects CNVs of as small a size asa few hundred BPs
aCGH is MAINLY used for postnatal diagnosis of complex phenotypes associated with ___ ___.mental retardation
aCGH can also be used in the prenatal field, but is limited here due to its inability to identify ___ ___ ___ and ___ with a cell line poorly represented (less than ___).balanced chromosomal rearrangements ; mosaicism ; (20%)
The test and reference DNAs are labelled by two different fluorescent substances, typically ___ and ___ (respectively).red ; green
The two labelled DNAs are incubated onto an ___, where there's a glass or plastic on which short ___ of ___, known as ___, are fixed.array ; fragments ; DNA ; probes
Each ___ in the array represents a specific ___ of the human ___.probe ; region ; genome
aCGH platforms can be divided into two main categories, depending on the type of probes fixed onto the array: ___ arrays and ___ arrays.BAC ; oligo (the former is "old generation"; the latter has smaller and more specific probes, giving higher resolution and accuracy)
Advantage over FISH/conventional karyotyping: The molecular karyotype can be used without a ___ clinical ___ or a ___ diagnosis, increasing the ___ of this test and the probability of correctly diagnosing pathologies associated with chromosomal abnormalities.specific ; diagnosis ; suspected ; applicability

Section 4: Restriction and PCR

Section 4.5: qPCR and qfPCR (advanced PCR)

Section 5: Gene scan; mRNA sequencing; Southern blotting

Question Answer
Southern blot analysis will reveal many aberrations (e.g. CNVs?) but will not always detect ___ ___ and is not ideal as a routine technique.small deletions
The key to Southern blotting is ___hybridization
Formation of a dsDNA molecule from a ssDNA ___ and a ss ___ DNA = hybridizationprobe ; target
In SB, after extracting the DNA from cells, it is ___ with ___ and then the ___ are separated by ___ according to ___.restricted ; enzymes ; fragments ; electrophoresis ; size
RFLP (restriction fragment length polymorphism)variation in restriction fragment lengths bwn individuals when cut by a restriction enzyme
RAPD (random amplified polymorphic DNA)primers with arbitrary sequences are used for amplification, so the amplified DNA segments are random (random DNA markers)
Use SB to identify ___ ___ in a DNA sample as well as to ___ desired DNA for ___ of rDNA.specific DNA ; isolate ; construction
Southern blotting can identify ___, ___ and gene ___.mutations ; deletions ; rearrangements
SB is used in prognosis of cancer and in ___ diagnosis of ___ ___.prenatal ; genetic diseases
Southern blotting is useful in DNA ___, e.g. for paternity or maternity testing, criminal and personal identification.fingerprinting

Section 6: MLPA (multiplex ligation-dependent probe amplification)

Question Answer
a multiplex ___ method detecting abnormal ___ ___ of up to 50 different genomic DNA or RNA sequences.PCR ; copy numbers
For most hereditary conditions, (partial) gene deletions or duplications account for ___ than ___% of all disease-causing mutations, BUT for many other disorders this is ___ to ___% or even higher still (i.e. MLPA is relevant in certain clinical settings).less ; 10 ; 10 to 30
Methods which were primarily developed for detecting point mutations, such as ___ and DHPLC, generally fail to detect ___ ___ changes.sequencing ; copy number (das why MLPA is good)
What's DHPLC, precious?Denaturing High Performance Liquid Chromatography *gollum* *gollum*
Compared to FISH, MLPA not only has the advantage of being a multiplex technique, but also one in which very ___ ___ (___to___ nt) are targeted, enabling it to identify the frequent, ___ ___ ___ which are too small to be detected by FISH.small sequences ; (50-70) ; single gene aberrations
Compared to aCGH, MLPA is a ___ ___ and technically ___ method.low cost ; uncomplicated
MLPA is not suitable forgenome-wide research screening
MLPA may be a good alternative to ___-based techniques for many routine applications.array
Applications ranging from the relatively ___ (DDS?) to the ___ ___ (hPAB)common (Duchenne, DiGeorge syndrome, SMA) ; very rare (hereditary pancreatitis, antithrombin deficiency, Birt-Hogg-Dube syndrome)

Section 7: Sequencing - background

Question Answer
STRP (short tandem repeat polymorphism)when homologous STR loci differ in the # of repeats bwn individuals
"ATTCG ATTCG ATTCG" is an example ofSTR (the sequence ATTCG is repeated 3 times; if in another individual it is repeated 5 times, this is STRP)
A 2-5 BP-long DNA motif that is repeated (typically _________-_________ times)microsatellite ; (5-50)
A minisatellite is a tract of repetitive DNA in which the DNA motif is _________-_________ BPs long and is repeated _________-_________ times.10-10 ; 5-50
T or F- Microsatellites have a higher mutation rate than other DNA areas.True. This contributes to high genetic diversity.
Micro- and minisatellites are together classified asvariable number of tandem repeats (VNTR)
Relationship bwn STRs and microsatellites?A STR is a kind of microsatellite
T or F- The repeating sequence in STRs varies bwn individuals, creating genetic diversity.False. The number of repeats is what varies; the sequence of BPs itself does not change from person to person.

Section 7.1: Sanger sequencing

Question Answer
First generation sequencing/Sanger sequencing used the ___ ___ method.chain termination
ddNTPs =ddATP, ddTTP, ddCTP, ddGTP
A type of deoxynucleoside triphosphate (dNTP) that lacks a 3′ hydroxyl group and has a hydrogen atom insteaddideoxynucleotide (ddNTP)
When the ddNTP bases bind to the growing DNA sequence, they ___ replication as they ___ ___ other bases.terminate ; cannot bind
To perform Sanger, you put the genetic information you want sequenced in a solution (with primers added), then ___ the solution into ___ ___ reactions, each containing a dNTP mix with ___ of the ___ ___ substituted with ddNTP.divide ; 4 PCR ; 1 ; 4 nucleotides
Disadvantages: Sanger is ___ and ___.slow ; expensive
Sanger sequencing also involves ___ the DNA samples on a ___ with ___ ___.running ; gel ; 4 lanes

Section 7.2: WES (whole exome sequencing)

Section 7.3: NGS - background

Question Answer
NGS is a ___, ___, and ___ genome analysis.rapid ; affordable ; accurate (also cost-effective)
NGS uses ___ ___ chain-terminating ddNTPs.fluorescently labelled (fluorochrome attached to nucleotide)
Unlike Sanger, NGS does not use ___ to separate the samples according to size, but rather uses the ___ ___.gel ; capillary electrophoresis
In NGS, the size of samples is measured by their ___, i.e. how ___ they are ___ towards the ___ ___.charge ; close ; pulled ; positive electrode (the closer they are/the more negative their charge, the LARGER the fragment is)
NGS is AKAhigh-throughput sequencing
NGS is a ___ term, describing a number of different modern sequencing technologies (SIRI):Illumina (Solexa) ; Roche 454 ; Ion torrent: Proton / PGM sequencing ; SOLiD
In NGS, vast numbers of ___ ___ are sequenced in a single stroke.short reads (so the input sample must be cleaved to shorter sections)
The length of the fragments into which the input sample must be cleaved before the actual sequencing starts, depends onthe particular sequencing machinery used (e.g. illumina vs ion torrent)

Section 7.4: NGS - Illumina (gene panels, medical exome, solid, ion torrent, illumina, 454, pacific bioscience, Oxford nanopore)

Question Answer
In Illumina, _________-__bp reads are used.100-150
In Illumina, the DNA fragments are ligated to ___ (looks like a stick) which are annealed to a slide.adaptors
In Illumina, first ___ is carried out to ___ each read, creating a spot on the slide with many ___ of the ___ ___.PCR ; amplify ; copies ; same read
After PCR is done, ___ ___ nucletodies and DNA polymerase are added to the slide; each one of the nucleotides has a ___, so that only ___ ___ is added at a time.fluorescently labelled ; terminator ; one base
After the first nucleotide is added and the signal is recorded, the ___ are ___ (to make the polymerase continue) and the fluorescent signal is ___ (to prevent contamination), and the next cycle beings.terminators ; removed ; removed
All of the sequence reads will be ___; the read ___ depends on the number of cycles carried out.the same length ; length

Section 7.5: NGS - 454

Question Answer
Compared with Illumina, Roche 454 can sequence-much longer reads (up to 1 kb, compared with Illumina's 150 bases)
Like Illumina, 454 sequences ___ reads ___ ___ by reading optical signals as bases are added.multiple ; at once
The adaptors in 454 are annealed to a ___, not a slide.bead (1 DNA fragment per bead)
Each bead is placed in a single ___ of a slide; so each ___ will contain a single bead covered in many PCR copies of a single sequence.well

Section 7.6: NGS - Ion torrent: Proton / PGM sequencing

Section 7.7: NGS -

Section 8: Other (hybridization-based strategies, singleplex and multiplex PCR, DNA target enrichment)