DNA MICROARRAY Flashcards
1
Q
STUDY OF HEREDITY
A
GENETICS
2
Q
It focuses primarily on the likelihood of _____________.
A
Genetics - developing cancer
3
Q
tests find ______, not diseases.
A
genetics - mutations
4
Q
STUDY OF GENES, HOW THEY INTERACT AND EXPRESSED AS A WHOLE
A
genomics
5
Q
If genetics is the study of ______, genomics is the study of _____ and how they interact, how they are passed on from generation to generation, and how they are expressed as a whole.
A
heredity, genes
6
Q
Genetics and genomics are _________ that focuses on cancer itself and can help determine:
A
profiling tools
- prognosis (how aggressive)
- prediction (what is likely the benefit)
7
Q
It is an international scientific research project.
A
HUMAN GENOMICS PROJECT
8
Q
HUMAN GENOMICS PROJECT
→ Determining the _______.
→ ldentifying and mapping all of the ______.
A
sequence of human DNA, human genes
9
Q
HUMAN GENOMICS PROJECT
Key Findings:
There are ________ genes in a human
being
A
approximately 20,500
10
Q
They are the basic physical and functional unit of heredity
A
GENES
11
Q
They are a part of a DNA.
A
GENES
12
Q
Some genes act as instructions to make molecules called _____
A
proteins
13
Q
Each human cell contains ___ chromosomes, and each chromosome contains _____ genes (based on the findings of the Human Genomic Project)
A
46
20,500
14
Q
2 TYPES OF GENES
A
Structural and Regulatory Genese
15
Q
A gene that codes for any RNA or protein product other than a regulatory factor.
A
structural genes
16
Q
Encode for proteins or RNA
A
structural genes
17
Q
Encode for mRNAs, rRNAS, and tRNAS.
A
structural genes
18
Q
Gene products have either structural or functional importance
A
structural genes
19
Q
A gene involved in controlling the expression of one or more other genes.
A
regulatory genes
20
Q
Encode for transcription factors or regulatory RNA
A
regulatory genes
21
Q
Regulate the expression of structural genes
• Regulatory genes process the ________.
• Ensures that the appropriate genes are __________
A
regulatory genes
- turning on and off of the gene
- expressed at the proper time.
22
Q
EXAMPLES FOR STRUCTURAL GENES
A
Lac Z,A,Y genes of lac operon, actin gene
23
Q
EXAMPLES OF REGULATORY GENES
A
Lac I gene, CAP gene
24
Q
WHOLE DNA GENE 2 PARTS
A
- Intron
2. Exon
25
portion of a gene that does not code for amino acids/ proteins
intron
26
- part of thw whole gene that are just intermissions
| - They are just there for the whole DNA to have time in forming a final mature RNA.
intron
27
portion of a gene that will form a part of the final mature RNA produced by that gene after introns have been removed by RNA splicing.
Exons
28
occurs only in a particular type of cell or
| tissue where only a subset of an organism's DNA will be expressed as mRNAs at any given time.
gene expression
29
The gene expression is focused on the _________ (________) where the 20,500 genes are activated simultaneously, and these genes are only activated when they are needed.
Central Dogma of Life, DNA -> RNA -> protein
30
The unique pattern of gene expression for a given cell or tissue is referred to as its __________.
*each gene has a unique pattern
molecular signature
31
If gene __ is activated (after the gene expression), it
| means the patient has cancer. (Indication of cancer)
gene B
32
Why is gene expression important in any
| malignancy/disease?
The use of gene expression profiling and development of gene biomarkers/signatures for cancer allows for the diagnosis, progression and aggressiveness analyses, prognosis, and prediction of therapeutic treatment.
33
MAIN TYPES OF GENE EXPRESSION ASSAYS
1. Serial Analysis of Gene Expression (SAGE)
2. Short Oligonucleotide Arrays SOA (Affymetrix)
3. Long Oligonucleotide Arrays LOA (Agilent Inkjet)
4. Fiber Optic Arrays FOA (Ilumina)
5. cDNA Arrays (Brown/ Botstein)
34
It does not use the whole sequence of genes; it only
| uses a small part of the gene.
Short Oligonucleotide Arrays (Affymetrix)
35
Father of Microarray Technology
Mark Schena
36
The slide cDNA microarray which was discovered by
| Brown and Botstein.
cDNA array
37
Each slide contains multiple spots, and each spot
| contains the DNA clone
cDNA array
38
The sample used in cDNA array is usually _______
PCR generated
39
Developed a scanner for reading the output
Steve Fooder
40
Developed a quantitative way of monitoring Gene Expression Patterns with complementary DNA Microarray.
Patrick Brown and Mark Schena
41
DNA Microarray is based on the ___________
Principle of Southern Blotting in the year 1975
42
The concept of DNA Microarray began in the ______.
mid-1980s
43
DNA microarray is also known as
DNA chip or Biochip
44
It is a technology that can sequence thousands of genes at a time in order to quantify the expression of the said genes
DNA Microarray
45
The bonding of probes and targets is referred to as
“HYBRIDIZATION”.
46
This is the DNA of a sample of interest which
| is compared to a reference DNA or 'control DNA'.
Target DNA
47
The gene that is placed into the hole in the
| glass slide.
probe
48
________ in general, are a snapshot that captures the
| activity pattern of thousands of genes at once.
microarray
49
a collection of features spatially arranged in a twodimensional grid, arranged in columns and rows.
the array/slide
50
The slide consists of a thousand to hundreds of thousands
| of spots per square inch
Array /slide
51
Each spot holds ______ of copies of a DNA sequence from one gene
millions
52
3 MAIN TYPES OF DNA MICROARRAY
1. Spotted array
2. In-situ array
3. Self-assembled array
53
→ These arrays use a poly-lysine coated microscope slide.
SPOTTED ARRAY
54
A robotic arm dips a small tip into DNA and then onto the glass surface to fluorescently label the sample
Spotted array
55
The probes need to be synthesized prior to chip preparation
spotted array
56
First arrays to be created
Spotted arrays
57
it is the most straightforward to use and relatively inexpensive.
spotted array
58
Allows the user to check the purity and quantity of the DNA or cDNA to be used
spotted array
59
can be time-consuming as the DNA or RNA have to be extracted, converted into cDNA (in the case of an RNA), and then purified.
Spotted array
60
These arrays use light and photolithography to create
| probes on chips.
In-Situ Array
61
These probes are oligonucleotides that match down sequences of targeted DNA
IN-SITU ARRAY
62
These arrays are faster than spotted arrays and do not need cDNA handling; it only requires the target sequence to be known.
In-Situ Array
63
- have higher sensitivity
| - are costly and require specialized machinery to synthesize many arrays themselves
In-Situ Array
64
These arrays are fiber optics.
Self-assembled array
65
DNA is assembled on small polystyrene beads.
SELF-ASSEMBLED ARRAY
66
Some versions contain wells already pre-made in a glass to hold the beads.
SELF-ASSEMBLED ARRAY
67
The beads are encoded with different combinations of fluorophore so that the randomly assembled oligos are identified by their specific combinations.
SELF-ASSEMBLED ARRAY
68
2 COMMON METHODS OF DNA MICROARRAY
1. Glass Slide cDNA microarray
| 2. DNA chip (oligonucleotide) Microarray
69
→ Conventional method.
Glass Slide cDNA Microarray
70
First type of DNA Microarray technology developed.
Glass slide cDNA Microarray
71
Invented by __________ & his colleagues at Stanford University.
Glass slide cDNA microarray- Patrick Brown
72
Uses a robotic device which deposits (spots) a nanoliter sample of DNA onto a coated microscopic glass slide (Spot size: 50-150 micrometer in diameter).
Glass slide cDNA microarray
73
The slide is usually made commercially and is made of glass/silicon/nylon.
Glass slide cDNA microarray
74
Each slide contains thousands of spots, and each spot contains a probe for a different gene
Glass slide cDNA microarray
75
→ Probes can attach to the slide by robotic means.
Glass slide cDNA microarray
76
ADVANTAGES:
• Relatively affordable with a ____ cost.
• It requires _______.
• Prior knowledge of sequence is _______.
• Good choice for ______ discovery.
• Necessary clone sets are available for numerous
organisms (_______)
Glass slide cDNA microarray
- lower
- no specialized equipment
- not necessary
- new gene
- humans, mice, rats
77
DISADVANTAGES
• Requires ______ for synthesizing & purifying DNA solutions before microarray fabrication.
• _______ of probe (Slide DNA) as compared to oligonucleotides array.
• _________ may occur due to common sequences from the same gene family
Glass slide cDNA microarray
- intensive labor
- Low density
- cross hybridization
78
______ are high-precision robots with metal pins that dip into DNA solution and taps down on the glass
slide.
Microarray spotters
79
Glass slides cDNA microarrays uses ______
• DNA spotting done by _____ arm using multiple pins.
• DNA in a ____ plate.
• DNA is usually _______.
• ______ can also be spotted.
spotting arrays
- robotic
- microtiter
- PCR amplified
- oligonucleotides
80
The process is done by prefabricating or synthesizing single-stranded oligo.
DNA Chip (Oligonucleotide) Microarray
81
Since oligos are usually ____, the density of these arrays is much higher.
short - DNA Chip (oligonucleotide) microarray
82
DNA Chip (Oligonucleotide) Microarray is traditionally called
'Gene Chips' (Gene Chip Array) by
| commercial suppliers, such as Affymetrix, Agilent
83
ADVANTAGES:
• Fast
• Specific
• Reproducible
DNA chip microarray
84
DISADVANTAGES
• In-situ oligonucleotide array formats tend to have _______ specialized equipment to carry out hybridization, staining of label, washing, and quantitation
process.
• Short sequences (oligonucleotides 10-15 base pair only) used on the array have __________ compared with glass cDNA microarrays.
```
DNA chip (oligonucleotide) Microarray
expensive, decreased sensitivity/binding
```
85
DNA MICROARRAY (Procedure)
1. Collection of samples
2. Isolation of mRNA
3. Creation of Labeled DNA
4. Hybridization
5. Microarray scanner
6. Analyzation of Data
86
COLLECTION OF SAMPLES
| Samples which can be collected:
a. peripheral blood
b. aspirate fluid
c. swab sample
d. lavages
- any cells that have DNA can act as sample
87
ISOLATION OF mRNA
- Extract the RNA from the samples. Using either a column or a solvent such as ________
- After isolating the RNA, we need to isolate the ____ from the rRNA and tRNA. The mRNA has a poly-A tail, so we can use a column containing beads with poly-T tails to bind with the mRNA.
- Rinse with _____ to release the mRNA from the beads. In turn, the buffer disrupts the Ph, disrupting hybrid bonds
phenol chloroform, mRNA, buffer
88
CREATION OF LABELLED DNA
• Add a labeling mix to the RNA. The labelling mix contains poly-T primers, reverse transcriptase (to make cDNA), and fluorescently dyed nucleotides
* Add Cyanine-3 (______) to the healthy cells.
* Add Cyanine-5 (______) to the cancerous cells.
fluoresces green, fluoresces red
