Kandpal lectures from 12 4 Flashcards
genomic cloning, cDNA cloning, library definitions
if chromosomal DNA is used as a starting material then it is termed as genomic cloning
–> clones will contain genes with introns and exons
cDNA cloning: single stranded mRNA–> double stranded cDNA by reverse transcriptase
–> clones will contain exon sequences–> which can be translated into proteins
-use mRNA and hybridize it with a poly (T) primer–> make DNA cope with reverse transcriptase –> degrade RNA with RNase and then synthesize a complementary DNA strand using DNA polymerase.
cDNAs can be cloned in special vectors for making proteins, hormones or vaccines
==> DNA will br cleaved with restriction nuclease and then the fragment will join via DNA ligase producing the recombinant DNA
library: Library is a collection of clones (vector + DNA/cDNA) of whole genome fragments or cDNAs corresponding to mRNAs.
southern hybridization, northern hybridization and western hybridization. steps for hybridization
Southern hybridization: electrophoresis of the digested DNA and hybridizing with a gene specific probe that contains a radioactive or fluorescent label (probe can be DNA or RNA).
–> DNA stained w/ethidium bromide and visualized under UV light–> transferred onto a nylon membrane–> membrane hybridized to a specific probe and then exposed to x-ray
northern hybridization: determines the Abundance of a specific mRNA in a tissue/cell->Target RNA is hybridized to a gene-specific RNA or DNA probe
–>electrophoresis of RNA isolated and transferred onto a nylon blot–> RNA blot is hybridized to a radioactive probe and exposed to x-ray
Western hybridization: determines the Abundance of a specific protein in a cell/tissue –>Total protein isolated from cell is electrophoresed and immobilized onto a membrane and then probed with an antibody.
–> protein separated by electrophoresis –> transfer protein onto membrane–> protein identified by antibody binding –> protein-antibody complex detected by color development or exposure to x-ray
Steps for hybridization
- Denaturation: Strands falling apart at high temperatures
- Renaturation: Complementary strand annealing
- Probe hybridization: Search of sequences (identical or related)
How does DNA sequencing work?
add normal deoxyribonucleoside triphosphate precursors (dATP, dCTP, dGTP, dTTP) and a small amount of one dideoxyribonucleoside triphosphate (ex. ddATP) to a single stranded molecule of DNA to be sequenced and a labeled primer
–> the incorporation of the ddATP will block polymerase–> terminating DNA synthesis
based on the lengths of each fragment for each dideoxyribonucleoside triphosphate, you can determine the DNA sequence
run all of the fragments onto a gel and DNA sequence can be read from the bottom up of the gel
you can also do the same thing using all 4 ddNTPs in one tube labeled with different colors==> then run them on a capillary gel and shoot a laser through them to detect which ddNTP is detected in what order–> sequence
gene knock-out
Mutant gene cloned into vector (gene fragment) and introduced in ES (embryonic stem) cells in a culture.
Let each cell grow and form colonies, and then test for the colony which the DNA fragment that was introduced (mutant) has replaced the one copy of the normal gene–> Homologous recombinants, where specific gene is replaced.
then inject these ES cells into the early embryo
Injection of engineered cells into mouse embryo.
Chimeric animals–> some native and some engineered ES
selected offspring will then breed to test for the gene in germ line
ex: FGF5, a negative regulator of hair growth, can be knocked out in mice–> producing long haired mice
Homologous Recombination
Homologous recombination is involved in
genetic re-assortment during gametogenesis.
DNA repair.
clearing replication hurdles.
Recombination can also lead to deleterious effects such as: deletions, inversions, translocations, and other rearrangements- some leading to cancers.
Chronic myeloid leukemia
have a small chromosome, Philadelphia (Ph).
Translocation joins the ABL oncogene on chromosome 9 with the 5’ part of BCR gene on chromosome 22
ABL (tyrosine kinase) gene is fused to BCR. The fusion gene becomes constitutively active. Overexpression of oncogene product (tyrosine kinase)–> MAP K and excessive proliferation of cells
gleevec, an inhibitor of Abl is used to treat CML
Burkitt’s lymphoma
In Burkitt’s lymphoma, MYC oncogene from chromosome 8 is translocated to IGH locus on chromosome 14.
Myc gene is placed in a chromatin domain that is highly active in B-cells, the antibody producing cells (IgH locus).–> up-regulated expression of MYC protein==> proliferation of B cells
no changes to MYC 2 or 3 exons but MYC 1 becomes noncoding
α-thalassemia (hemoglobinopathy)
Chromosome 16 has two copies of normal α-globin genes, a pseudo α-globin and many blocks of repetitive DNA. Unequal crossovers between misaligned X- or Z-repeats can produce chromosomes with 1-3 active α- or no active α-globin. • 4 copies = normal. • 3 copies = silent carrier. • 2 copies = α-thalassemia. • 1 copy = HbH disease. • 0 copies = Hydrops Fetallis.
Stages of prophase in meiosis I
Five stages of prophase in meiosis-I leptotene zygotene pachytene diplotene dikinesis
Recombination (synaptonemal complex at zygotene stage; crossing-over at pachytene)
Recombination and gene mapping
Recombination is a measure of separation between two loci.
closer two loci are, the more likely they are to be linked==> less chance of recombination
2 loci far apart==> more likely recombination
if a disease travels with a specific marker allele, the disease causing the mutation is linked to the marker
- -> the frequency of recombination allows for an estimate of the distance from the marker
- infrequent recombination==> shorter distance
- frequent recombination==> further apart
1% recombinants=1 centiMorgan
X-linked dominant pedigree
if a male is affected, all of the males daughters will be affected and none of his sons
children of an affected mom have a 50% chance of being infected (M or F)
Y-linked pedigree
ALL sons of affected males are affected
von Reclinghausen disease (neurofibromatosis 1) symptoms and how linkage was determined
Autosomal dominant
Brwonish spots on skin (café au lait)
Benign nodules on iris
Neurofibromas (fleshy benign tumors along the course of nerves)
Serious growth abnormalities and deformities
Linked to chromosome 17
Restriction fragment length polymorphism (RFLP) markers were used.
RFLP marker sequences are either susceptible or resistant to digestion with specific restriction enzymes. mutations.
digest chromosomal DNA with EcoRI (restriction enzymes), run on gel, blot and hybridize with a probe
- Specific restriction enzymes digest at certain sites creating a certain length fragment (3 kD and 1.7 kD in WT).
- Mutations can disrupt restriction sites and not be cleaved, creating larger fragments (4.7 kD).
compare blot to pedigree to see if mutation is in affected individuals
LOD Score
logarithm of ratio of liklihoods of linkages
If the ratio is 3 i.e. odds are 1000 times in favor of linkage–>Two loci are linked
Severe Combined Immuno deficiency (SCID)
Caused by impaired functions of B- and T-cells.
Patients are unable to mount any immune response. Could result in life-threatening infections.
Autosomal recessive SCID patients have adenosine deaminase deficiency.
X-chromosome linked SCID is most common. X-SCID is due to IL2R deficiency. Bone marrow CD34+ cells from X-SCID are treated ex vivo for correcting the IL2R deficiency.==> CD34+ stem cells are removed and injected with retrovrial vectors carrying the cytokine receptor gene, and allowed to multiply==> reinserted in the pt.
