Chromosome structure
#Cytogenetics
The study of inheritance in relation to the structure and function of chromosomes. It analyzes the number and structure of human and animal chromosomes
Human has 46 chromosomes.
Cytogenetic analyses are commonly performed during pregnancy to determine if a fetus is at risk for common aneuploidies (syndromes caused by having extra or missing chromosomes), syndromes caused by structural abnormalities (like unbalanced translocations or inversions), or to determine if extra or missing genetic material is present through cytogenetic microarray testing.
Trisomy are 3 copies of certain chromosomes.
Cytogenetic analyses can be performed on a newborn or child with multiple anomalies or developmental delays to look for a potential chromosomal abnormality.
Chromosome analysis of embryonic stem cells and induced pluripotent stem (iPS) cells is necessary
classical cytogenetic procedures (such as G-banding) and advanced molecular techniques such as genomic microarray analysis.
Karyotype is a test to identify and evaluate the size, shape, and number of chromosomes in a sample of body cells. Extra or missing chromosomes, or abnormal positions of chromosome pieces, can cause problems with a person's growth, development, and body functions.
G-banding, G banding, or Giemsa banding is a technique used in cytogenetics to produce a visible karyotype by staining condensed chromosomes
#Immunohistochemistry (IHC)
Its a process of selectively imaging antigens (e.g. proteins) in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues.
e.g. Staining process performed on fresh or frozen breast cancer tissue removed during biopsy. IHC is used to show whether or not the cancer cells have HER2 receptors and/or hormone receptors on their surface, The IHC can be used to screen the HER-2 status.
IHC protocol steps:
(1) Fixation – to keep everything in its place
(2) Antigen retrieval – to increase availability of proteins for detection
(3) Blocking – to minimize background
(4) Antibody labeling and visualization
For peroxidase-based detection systems, always use a peroxidase-blocking step.
#FISH (fluorescent in situ hybridization assay)
Its a molecular cytogenetic technique that uses fluorescent probes that bind to only those parts of the chromosome with a high degree of sequence complementarity.
To carry out FISH, scientists first unzip the DNA that makes up both the tagged gene and the chromosomes into two separate strands.A commercially available double-color probe
FISH is considered as a gold standard because of its sensitivity and specificity. FISH was approved by the US Food and Drug Administration (FDA) as the standard method for the detection of ALK gene (encoding ALK receptor tyrosine kinase) rearrangement in clinical practice.
*IHC and FISH can tell about the assessment for Her-2 status in breast cancer
The concordance rate between IHC and FISH is controversial
It may due to the different sensitivity and specificity of the antibodies and probe used in this study
A total concordance of 82.0% was observed
#Chromogenic in situ hybridisation (CISH)
It is a cytogenetic technique that combines the chromogenic signal detection method of IHC techniques with in situ hybridization
#Polymerase chain reaction (PCR)/ Real-time polymerase chain reaction
A real-time polymerase chain reaction (Real-Time PCR), also known as quantitative polymerase chain reaction (qPCR)is a modification of PCR, which monitors the amplification of a targeted DNA molecule during the PCR, i.e. in real-time.
Reverse transcription PCR (RT-qPCR) is used when the starting material is RNA. In this method, RNA is first transcribed into complementary DNA (cDNA) by reverse transcriptase from total RNA or messenger RNA (mRNA). The cDNA is then used as the template for the qPCR reaction. The reverse transcriptase allows a single strand of RNA to be translated into a complementary strand of DNA.
Denaturing – when the double-stranded template DNA is heated to separate it into two single strands.
Annealing – when the temperature is lowered to enable the DNA primers to attach to the template DNA.
Extending – when the temperature is raised and the new strand of DNA is made by the Taq polymerase enzyme.
#DNA microarray
DNA microarray (also commonly known as DNA chip or biochip) is a collection of microscopic DNA spots, attached to a solid surface. Its used to measure the expression levels of large numbers of genes simultaneously or to genotype multiple regions of a genome i.e parallel analysis of genetic material in the genome. Its called gene expression profiling, which finds the cluster of genes turned on or off.
Usage: for high-resolution HER2 testing; to study insecticide resistance in African malaria-carrying mosquitoes.
Each DNA spot contains picomoles (10−12 moles) of a specific DNA sequence, known as probes (or reporters or oligos). These can be a short section of a gene or other DNA element that are used to hybridize a cDNA or cRNA (also called anti-sense RNA). Probe-target hybridization is detected and quantified. A high number of complementary base pairs in a nucleotide sequence means tighter non-covalent bonding between the two strands. After washing off non-specific bonding sequences, only strongly paired strands will remain hybridized. DNA microarrays can be used to detect DNA or detect RNA.
The process of measuring gene expression via cDNA is called expression analysis or expression profiling
control and diseases sample
extract mRNA
reverse transcription/ make cDNA
fluorescent labelling/tags (green for normal DNA, red for diseased DNA)
Combine equal amount of these labels on the spots containing probes on microarray plate
let hybridization occur (Affymetrix GeneChip platform)
(The precise location and sequence of each spot is recorded in a computer database.)
scan with laser light(check the pattern of red and green or yellow (both types of genes in equal amount); black is constitutive expression)
To capture regions of interest
biotin-labeled RNA 'bait' probes are used to 'fish' specific DNA sequences out of a 'pond' of DNA fragments. When the RNA is hybridized to a fragmented genomic library, DNA and RNA hybrid duplexes are formed, which can be collected and captured using streptavidin beads, whereas the uncaptured, nontargeted portion is thrown away during washing. In an elution step, DNA is then released from the beads and RNA is digested.
==============
(https://geneed.nlm.nih.gov/topic_subtopic.php?tid=15&sid=17)
#Cytogenetics
The study of inheritance in relation to the structure and function of chromosomes. It analyzes the number and structure of human and animal chromosomes
Human has 46 chromosomes.
(https://ghr.nlm.nih.gov/primer/basics/howmanychromosomes)
Chromosomal abnormalities can happen when egg and sperm cells are being made, during early fetal development, or after birth in any cell in the body. Changes to chromosome structure can disrupt genes, causing the proteins made from disrupted genes to be missing or faulty. Depending on size, location, and timing, structural changes in chromosomes can lead to birth defects, syndromes or even cancer.Cytogenetic analyses are commonly performed during pregnancy to determine if a fetus is at risk for common aneuploidies (syndromes caused by having extra or missing chromosomes), syndromes caused by structural abnormalities (like unbalanced translocations or inversions), or to determine if extra or missing genetic material is present through cytogenetic microarray testing.
Trisomy are 3 copies of certain chromosomes.
Cytogenetic analyses can be performed on a newborn or child with multiple anomalies or developmental delays to look for a potential chromosomal abnormality.
Chromosome analysis of embryonic stem cells and induced pluripotent stem (iPS) cells is necessary
classical cytogenetic procedures (such as G-banding) and advanced molecular techniques such as genomic microarray analysis.
Karyotype is a test to identify and evaluate the size, shape, and number of chromosomes in a sample of body cells. Extra or missing chromosomes, or abnormal positions of chromosome pieces, can cause problems with a person's growth, development, and body functions.
G-banding, G banding, or Giemsa banding is a technique used in cytogenetics to produce a visible karyotype by staining condensed chromosomes
Its a process of selectively imaging antigens (e.g. proteins) in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues.
e.g. Staining process performed on fresh or frozen breast cancer tissue removed during biopsy. IHC is used to show whether or not the cancer cells have HER2 receptors and/or hormone receptors on their surface, The IHC can be used to screen the HER-2 status.
IHC protocol steps:
(1) Fixation – to keep everything in its place
(2) Antigen retrieval – to increase availability of proteins for detection
(3) Blocking – to minimize background
(4) Antibody labeling and visualization
For peroxidase-based detection systems, always use a peroxidase-blocking step.
(https://rockland-inc.com/ihc-products.aspx)
#FISH (fluorescent in situ hybridization assay)
Its a molecular cytogenetic technique that uses fluorescent probes that bind to only those parts of the chromosome with a high degree of sequence complementarity.
To carry out FISH, scientists first unzip the DNA that makes up both the tagged gene and the chromosomes into two separate strands.A commercially available double-color probe
FISH is considered as a gold standard because of its sensitivity and specificity. FISH was approved by the US Food and Drug Administration (FDA) as the standard method for the detection of ALK gene (encoding ALK receptor tyrosine kinase) rearrangement in clinical practice.
(https://www.cureus.com/articles/7529-application-of-fluorescence-in-situ-hybridization-fish-technique-for-the-detection-of-genetic-aberration-in-medical-science)
FISH analysis with probes targeting the X chromosome can reveal a duplication of the X centromere.*IHC and FISH can tell about the assessment for Her-2 status in breast cancer
The concordance rate between IHC and FISH is controversial
It may due to the different sensitivity and specificity of the antibodies and probe used in this study
A total concordance of 82.0% was observed
#Chromogenic in situ hybridisation (CISH)
It is a cytogenetic technique that combines the chromogenic signal detection method of IHC techniques with in situ hybridization
#Polymerase chain reaction (PCR)/ Real-time polymerase chain reaction
A real-time polymerase chain reaction (Real-Time PCR), also known as quantitative polymerase chain reaction (qPCR)is a modification of PCR, which monitors the amplification of a targeted DNA molecule during the PCR, i.e. in real-time.
Reverse transcription PCR (RT-qPCR) is used when the starting material is RNA. In this method, RNA is first transcribed into complementary DNA (cDNA) by reverse transcriptase from total RNA or messenger RNA (mRNA). The cDNA is then used as the template for the qPCR reaction. The reverse transcriptase allows a single strand of RNA to be translated into a complementary strand of DNA.
(https://www.thermofisher.com/us/en/home/brands/thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-library/basic-principles-rt-qpcr.html)
There are three main stages of PCR:Denaturing – when the double-stranded template DNA is heated to separate it into two single strands.
Annealing – when the temperature is lowered to enable the DNA primers to attach to the template DNA.
Extending – when the temperature is raised and the new strand of DNA is made by the Taq polymerase enzyme.
(https://www.yourgenome.org/facts/what-is-pcr-polymerase-chain-reaction)
Since, DNA extraction is often required before PCR, teh steps of DNA extraction has been mentioned below. The three basic steps of DNA extraction are 1) lysis, 2) precipitation, and 3) purification.
(http://www.gentaurpromo.com/dna_rna_extraction/Products254b.html?pid=155&pkid=9&pttype=57)
(http://www.clontech.com/CN/Support/Applications/Nucleic_Acid_Purification/DNA_From_Soil)
Nowadays kits are used for DNA extraction...............
To extract DNA, tissue samples are taken from plants, and ground to break open the cells. Next, a buffered salt water solution is added into which DNA dissolves easily. CTAB buffer (cationic detergent ) is used to lyse plant cells whereas SDS (anionic detergent ) is used to lyse bacterial cells.
Detergents bind lipid molecules from plasma membranes to form micelles and these micelles are easily “washable” using water.
DNA microarray (also commonly known as DNA chip or biochip) is a collection of microscopic DNA spots, attached to a solid surface. Its used to measure the expression levels of large numbers of genes simultaneously or to genotype multiple regions of a genome i.e parallel analysis of genetic material in the genome. Its called gene expression profiling, which finds the cluster of genes turned on or off.
Usage: for high-resolution HER2 testing; to study insecticide resistance in African malaria-carrying mosquitoes.
Each DNA spot contains picomoles (10−12 moles) of a specific DNA sequence, known as probes (or reporters or oligos). These can be a short section of a gene or other DNA element that are used to hybridize a cDNA or cRNA (also called anti-sense RNA). Probe-target hybridization is detected and quantified. A high number of complementary base pairs in a nucleotide sequence means tighter non-covalent bonding between the two strands. After washing off non-specific bonding sequences, only strongly paired strands will remain hybridized. DNA microarrays can be used to detect DNA or detect RNA.
( https://www.genome.gov/10000533/dna-microarray-technology/)
Uses hybridization to monitor DNA/RNA abundance on a genome.
Cell -----> extract RNA -----> reverse transcription (purification and labelling by IVT)----->fragmentation (heat, Mg++) ----->hybridization-----> laser scanning ----->gene expression-----> ratio analysis
miRNAs plays role in normal physiology, also diseases like cancer. Hybridization based microarray technology has been used for miRNA profiling, but is hindered by its narrow detection range, more susceptibility to technical variation, and lack of ability to characterize novel miRNAs and sequence variation.
control and diseases sample
extract mRNA
reverse transcription/ make cDNA
fluorescent labelling/tags (green for normal DNA, red for diseased DNA)
Combine equal amount of these labels on the spots containing probes on microarray plate
let hybridization occur (Affymetrix GeneChip platform)
(The precise location and sequence of each spot is recorded in a computer database.)
scan with laser light(check the pattern of red and green or yellow (both types of genes in equal amount); black is constitutive expression)
To capture regions of interest
biotin-labeled RNA 'bait' probes are used to 'fish' specific DNA sequences out of a 'pond' of DNA fragments. When the RNA is hybridized to a fragmented genomic library, DNA and RNA hybrid duplexes are formed, which can be collected and captured using streptavidin beads, whereas the uncaptured, nontargeted portion is thrown away during washing. In an elution step, DNA is then released from the beads and RNA is digested.
==============
Mitogens: phytohemagglutinin, lipopolysaccharide (to increase the efficiency of EBV (Epstein-Barr virus)-mediated immortalization)
(http://tbl.med.yale.edu/cell_growth_control_2020/reading.php)
Immunosuppressive agents: cyclosporin A (to inhibit T cell-mediated killing of infected B cells)
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