Alkaline Phosphatase (AP

 Alkaline Phosphatase (AP)


Alkaline Phosphatase is an important tool in molecular biological processes like cloning. It removes 3’- phosphate groups from a variety of substrates. Although in laboratory, it is used to catalyze the removal of terminal 5’-(P), residues from single stranded or double stranded DNA and RNA. The resulting 5’-OH termini can no longer take part in ligation reactions, thus prevents self religation of vectors, reducing the background of transformed bacterial colonies that carry empty plasmids. This enzyme works optimally at alkaline pH (range of 89 in the presence of low Zn+2 concentrations) and hence derived the name.  Alkaline Phosphatase is isolated from various sources:-

 a) Bacterial Alkaline phosphatase Secreted in monomeric form into the Periplasmic space of E.coli, where it form dimers and gets catalytically activated. It’s a remarkably stable enzyme and is resistant to inactivation by heat and detergent. Thus, bacterial alkaline phosphatase is the most difficult to destroy in the reaction mix.

b) Calf Intestinal Phosphatase  Calf intestinal phosphatase is a dimeric glycoprotein isolated from bovine intestine. This has much more practical significance than bacterial alkaline phosphatase, since it can be readily inactivated from the reaction mixture using proteinase K or by heating at 650C for 30 minutes or 750C for 15 minutes in the presence of 10mM EGTA.

c) Shrimp alkaline phosphatase Extracted from cold water shrimp, can be inactivated readily by heating at 650C for 15 min. 

what is Terminal Deoxy Nucleotidyl Transferase?

what is Terminal Deoxy Nucleotidyl Transferase ?

Terminal transferase is an unusual DNA polymerase found only in prelymphocytes and in early staqes of lymphoid differentiation. Synthesis of single stranded tails at the 3’ ends of either single stranded DNA or double stranded DNA with protruding 3’ termini, by the enzyme Terminal Deoxy Nucleotidyl Transferase, is called tailing, can be used to generate protruding ends of defined sequence to facilitate cloning of fragments. It can be used to generate protruding ends of defined sequence, e.g poly A tails on the 3’ ends of the DNA insert and poly T tails on 3’ ends of the vector. Thus, the protruding ends of the DNA insert and vector will base pair under appropriate annealing conditions. Mg2+ cation is preferred when the nucleotide to be added is a purine while Co2+ is preferred for the addition of pyrimidines. The enzyme strongly prefers DNA with protruding 3’ terminus, although blunt or recessed 3’ termini are also used, but less efficiently, in buffers of low ionic strength with Co2+, Mg2+ or Mn2+ as bivalent cations. As many as thousands of deoxynucleotides can be incorporated using this enzyme on a template of DNA. Single nucleotide can be added to the 3’ termini of DNA if modified bases like dideoxynucleotides or cordycepin triphosphates are used instead of natural deoxynucleotide triphosphates.

What is Screening, Genetic Tests and Counseling of Cancer?

What is Screening, Genetic Tests and Counseling of Cancer?

Early diagnosis of cancer greatly increases survival; therefore, regular exams for cancer can help to prevent deaths from cancer. These include mammograms and Pap tests for women, prostate cancer tests for men, colonoscopy exams for colon cancer, and regular physical exams for other types of cancer. Individuals with a strong family history of cancer should consider genetic tests for cancer and cancer risk counseling. The focus of cancer risk counseling is the individual’s personal risk of developing cancer and appropriate actions based on that risk. The discovery of the BRCA1and BRCA2genes associated with early development of breast cancer has allowed women with a family history of early breast cancer to be tested for mutations in these genes. Only five to ten percent of breast cancers show evidence of inheritance. Of these, forty-five percent are associated with a mutation in BRCA1and thirty-five percent with BRCA2. The gene or genes for the remaining twenty percent are not yet known. If the BRCA1 and BRCA2test results are negative, there is no evidence that the woman will have breast cancer because of these mutations. However, she may get breast cancer because of somatic mutations in these or other genes. If the BRCA1 orBRCA2test is positive, other family members may be tested to determine whether the gene was inherited. If other family members are negative, then there is less chance of hereditary risk of this form of cancer, although the individual with the mutation does carry an increased risk of the disease. If the test is positive in other family members, there is an increased hereditary risk for breast cancer in that family. The absence of hereditary risk does not mean that there is no other risk for breast cancer. Decisions based on genetic tests can be very complicated. Individuals must be fully informed about the risks before they can make reasonable decisions. Genetic counselors are trained to help individuals make difficult decisions based on genetic tests. The cumulative risk of breast cancer to age seventy for a woman with a BRCA1mutation is about fifty-seven to eighty-five percent depending on whether she is in a high-risk family. Some women find the fear of cancer so disruptive to their lives that they choose mastectomy to prevent cancer. (This is called prophylactic mastectomy.) Similarly, women with BRCA1have a high lifetime risk of ovarian cancer, causing some of them to choose to have their ovaries removed. While these are difficult decisions, the availability of genetic information provides individuals with information that they can use to make such important medical decisions. A young woman with a strong family history of ovarian cancer might find by genetic testing that she does not have the BRCA1 mutation and should not consider removal of her ovaries.

What is Follicle in Human fertilization? Development if follicle.

What is Follicle in  Human fertilization? Development if follicle.

Follicle cells (from sex cords) surround the Primary oocyte.

The Follicle is the oocyteplus follicle cells

Primordial follicle -follicle cellspartially surround oocyte

Primary follicle –follicle cells form a complete layer Follicle cells form gap junctions with the oocyteand produce Meiotic inhibitory factor.
Follicle cells are called granulosacells

Granulosacell  layer enclosed by the membranagranulosa, a basement membrane that acts as a barrier to capillaries.

Zona pellucidasecreted by oocyteand
follicle cells –with microvillarconnections between the two. 

Ovary cells form 2 more layers –theca interna, theca external.

Follicle Development

Secondary follicle –formation of the antrum(cavity) fluid filled, liquor folliculi Hormone production, androgens and estrogen.

Tertiary or Graffianfollicle–12 hours prior to ovulation.
cumulus oophorus= mound of cells that house the secondary oocyte.

Oogenesiscontrolled by cycles (Menstrual) of hormone release: Hypothalamus to gonadotropinreleasing hormone(GnRH) Anterior to  pituitary Gonadotropins, includesluteinizing hormone(LH)
and Follicle stimulating hormone(FSH)

Ovulation -tertiary follicle protrudes like a blister on the surface of the ovary –then bursts in response to LH and FSH.

Corpus Luteum–Follicle after ovulation –hormone producing Progessterone.

What is Bacteriophage Artificial Chromosomes (BACs) ?

What is Bacteriophage Artificial Chromosomes (BACs) ?

These plasmids are circular DNA molecules carrying conventional antibiotic resistance marker, origin of replication derived from the F factor of E.coli, an ATP driven helicase (repE) to facilitate DNA replication and three loci (parA, parB and parC) for proper partitioning of the plasmid to daughter cells. BAC vectors have no packing constraints and there is no fixed limit to the size of genomic DNA that they accept. Usually the size of DNA is approximately 120-kilo base pairs.  

what is Shuttle vector in gene cloning ?

What is Shuttle vector in gene cloning ?

Cloning of foreign DNA is usually carried out primarily in E.coli since the organism is most thoroughly studied. But subsequent work often requires the foreign segment to be delivered to different host cells like eukaryotes. A number of vectors are devised to satisfy this requirement. These vectors are termed as shuttle vectors. These vectors have origins of replication of various hosts. The also contain fragments of eukaryotic viruses to facilitate entry into the cell or expression or integration in the cell itself. Thus shuttle vectors allow DNA to be transferred between two different species where it can be propagated by utilizing both the origins of replication. Usually the origins of replication are derived from bacterial and eukaryotic systems. Shuttle vectors also carry antibiotic resistance genes, which are functional in eukaryotes e.g. Neomycin (G418), Hygromycin, Methotrexate etc. All the DNA manipulation and characterization are done in prokaryotic system and then the manipulated DNA is introduced into the eukaryotic systems for protein expression and functional analysis. Eukaryotic host systems are better for expression of protein for few reasons:

1. Proper folding of the protein to attain functional activity .

2. Posttranslational modification of proteins for which prokaryotes does not possess any machinery. The most conventional and convenient model system for expression of eukaryotic proteins is yeast, Pichia pastoris, which is both genetically and physiologically well characterized. 

Yeast Artificial Chromosome (YACs)

Yeast Artificial Chromosome (YACs)

These are linear DNA molecules similar to yeast chromosome. Recombinant YACs are made by ligating large fragments of genomic DNA and then the resultant plasmid is introduced into yeast by transformation. The vector carries selection marker, DNA sequences called as telomere, so that the product can be stabilized inside the yeast cell, an origin of replication called autonomous replication origin, ARS. Large size of DNA can be inserted into YAC vectors, usually between 250kilobases to 400kilobasepairs. Large size of mammalian genomic libraries is also made with approximately 1 megabasepairs of foreign inserts. Insertion of foreign DNA into the cloning site inactivates a mutant expressed in vector DNA and formation of red rather than white colonies by yeast strain is observed. Thus transformants are identified as red colonies, which grow in yeast that is mutant for TRP1 and URA3, which ensure that the cell has received an artificial chromosome and with both the telomeres since it is complimented for both the mutations. And the colony also contains foreign DNA because it is red in color.