Lecture 4 Flashcards
Karyotyping
Chromosome imaging
Composition of a Chromosome
Centromere, p and q arm
Chromatid
Chromosome during replication or meiosis
First step for karyotyping
Using mitotic inhibitor
Second step for karyotyping
Lymphocytes are harvested and treated with hypotonic solution
Third step for karyotyping
Chromosomes are fixed and dropped onto glass
Fourth step for karyotyping
Chromosomes are stained and banding patterns is produced
Fourth step for karyotyping
Chromosomes are stained and banding patterns is produced
Types of chromsomes’morphology
Metacentric, Submetacentric and acrocentric
Tiny DNA of acrocentric chromosome
Satellite stalks
Incorrect number of chromosomes
Aneuploidy
One reason for reciprocal translocation
Two non homologous chromosomes have double strand breaks + NHEJ
Example of a reciprocal translocation of chromosomes (leading to Chronic Myeloid Leukemia)
Philadelphia chromosome (22)
FISH acronym meaning
Fluorescence In Situ Hybridization
For what metaphase FISH is used
Micro-deletion, micro-insertion, structural rearrangement
Name of technique used to detect DNA
Southern blot
Name of technique used to detect RNA
Northern blot
Name of technique used to detect proteins
Western blot
What does restriction enzyme do?
Cut DNA into smaller fragments
Three types of restriction enzymes
Blunt ends, 5’ sticky ends, 3’ sticky ends
How does Southern Blot + Restriction enzymes work?
Restriction enzyme cut after a specific sequence. If not present, not cut will be there and southern blot will detect longer dna
What does PCR stands for?
Polymerase Chain Reaction
Steps of PCR
Denaturation of DNA, primer annealing, DNA synthesis
Promoter
Sequence of a gene where RNA polymerase fixes before starting transcription
What molecule is used to sequence RNA?
Using cDNA
What does IP stand for
Immunoprecipitation
What is immunoprecipitation
Technique of precipitating a protein antigen out of solution using an antibody that specifically binds to that particular protein
What is Co-IP?
Selects an antibody that targets a known protein that is believed to be a member of a larger complex of proteins
What does CGH (microarray) stand for?
Comparative Genomic Hybridization
Limitations of array technology
- Can’t detect balanced chromosomal rearrangements (reciprocal translocations, inversions, polyploidy)
- Do not cover highly repeat-rich sequences
Steps of Sanger sequencing
- PCR
- Chop DNA
- DNA with nucleotides and (small amount) dideoxynucleotide (stop DNA chain elongation)
- Reading of dye-labelled fluorescent tags on ddNucl
What is NGS?
Next Generation Sequencing: Simultaneous sequencing of hundreds of millions of short sequences
Sequence detection technologies
- Sanger sequencing
- 454 pyrosequencing
- Illumina (Solexa) Sequencing
- SOLiD sequencing
- Ion Torrent sequencing
What is Bridge PCR?
72/94
Construction of recombinant DNA molecules in vitro
- Cleave vector and insert DNAs with compatibles sticky ends
- Ligation of DNA pieces
- Bacterial transformation => Bacteria are mixed with the recombinant plasmid and will inherit this recombinant DNA
How do we select only the bacteria with DNA inserted?
DNA inserted has also gene for penicilin resistance => Only these are alive
What is a library
A library is a way to store genetic information or a way to do genetic screening
What contain a cDNA library
It contains the expressed (or transcribed) genes of a given tissue
What is the main way to create a library?
Use of Bacterial Artificial Chromosome
What is BAC?
Bacterial Artificial Chromosome
What are the characteristics of a BAC?
- Vector of choice for current genomic library construction and screening
- Holds 100 to 200kb
- Stably propagated in E. coli
What are the two genetic approaches in model systems?
- Forward genetics: Which genes are responsible for a given phenotype
- Reverse genetics: What change in the phenotype is observed when a gene is altered
Approach to do Forward genetics
- Create genetic diversity
- Select the cells/organisms with the interesting phenotypes
- Identify genes involved
