mutations Flashcards
- Definition of cytogenetic clone
● - Minimum of two mitotic cells with gain of the same chromosome or structural abnormalities
● - Three mitotic cells with loss of same chromosome
- Robertsonian translocation (ROB)
Is a rare form of chromosomal rearrangement that in humans occurs in the five acrocentric chromosome pairs, namely 13, 14, 15, 21, and 22.
- Three steps of PCR reaction
● Denaturation- DNA heated to 95 C to make it single stranded
● Annealing- primer binds to complementary strand
● Primer extension - DNA polymerase extends primer
- List 4 test that may help in proving the clonality of cells
Flow cytometry
- FISH
- Cytogenetics
- PCR/RT-PCR
- T-cell clonality studies with T-cell receptor
- B Cell clonality studies
- Immunohistochemistry (Kappa/Lambda)
a. Missense mutation
○ Point mutation in which a single nucleotide change results in a codon that codes for a different amino acid
b. Nonsense mutation
○ A point mutation in a sequence of DNA that results in a premature stop codon leading to truncated, incomplete and usually nonfunctional protein product
○ There are three RNA stop codons: UAG, UAA, and UGA.
Copy-neutral loss of heterozygosity (CN-LOH)
(CN-LOH) is a genetic alteration that occurs when **one of two homologous chromosomal regions **is lost, but various mechanisms have ensured the presence of two identical copies of such region in the genome1
c. What is cDNA?
○ Complementary DNA = DNA synthesized from a single-strand RNA template in a reaction catalyzed by the enzyme reverse transcriptase
- Next generation sequencing
a. What is the principle of testing
● Massively sequencing millions of small fragments of DNA in parallel.
● Mapping the individual reads to the human reference genome through bioinformatics.
● Each of the three billion bases in the human genome is sequenced multiple times, providing high depth to deliver accurate data.
● NGS can be used to sequence:
○ Entire genomes
○ Specific areas of interest:
● NGS can be used to sequence:
○ Entire genomes
○ Specific areas of interest:
■ 22 000 coding genes (a whole exome).
■ Small numbers of individual genes.
What are the pros of NGS versus sanger sequencing
**Pros: **
● Improved accuracy and speed, and reduced manpower and cost.
● Captures a broader spectrum of mutations than Sanger sequencing
● Can discover novel mutations and disease causing genes.
● Increased sensitivity: detection of mosaic mutations, fetal DNA from maternal blood, or MRD
What are the cons of NGS versus sanger sequencing?
What is the problem in data analysis and interpretation
Cons:
● Not widely available
● Some regions sequence poorly or map erroneously due to extreme guanine/cytosine (GC) content or repeat architecture.
● Require special infrastructure, such as computer capacity and storage
● Requires high personnel expertise to comprehensively analyse and interpret the data.
● You need to batch sample to make it more cost effective
-You need very skillful and experienced personnel to manage and interpret the data. Some novel mutations are of unknown significance and you don’t know what to do with them!
Define the following (4.5 marks)
a. Telomerase
b. cosmid
c. ligase
a. Telomerase
Is a ribonucleoprotein that adds a species-dependent telomere repeat sequence to the 3’ end of telomeres, thus protecting the end of the chromosome from DNA damage or from fusion with neighbouring chromosomes.
b. cosmid
A plasmid into which a short nucleotide sequence of a bacteriophage has been inserted to create a vector capable of cloning large fragments of DNA
c. ligase
an enzyme that brings about ligation of DNA or another substance
What is the “digital library in NGS
NGS library preparation begins with the fragmentation of target DNA (genomic or cDNA).
This is followed by ligation of the DNA fragments to oligonucleotides
The ligated fragments are amplified by PCR; ideally, all sequences are represented many times in overlapping PCR products.
After sequencing, the reads from the library are aligned and assembled
in ddPCR, the PCR reactions occur simultaneously in droplets
