Recombinant human insulin is used in diabetes; growth hormone in growth hormone deficiency; erythropoietin in anaemia; interferon-alpha in some viral infections and cancers; interferon-beta in multiple sclerosis; factors VIII and IX in haemophilia A and B; tissue plasminogen activator in clot dissolution; hepatitis B surface antigen in vaccination; and G-CSF to stimulate neutrophil production after chemotherapy.
Examples include recombinant human insulin, growth hormone, erythropoietin, interferon-alpha, interferon-beta, factor VIII, factor IX, tissue plasminogen activator, hepatitis B vaccine antigen and granulocyte colony-stimulating factor.
EcoRI recognises the palindromic sequence 5'-GAATTC-3'. It cuts as 5'-G/AATTC-3' and 3'-CTTAA/G-5'. The products are DNA fragments with complementary single-stranded sticky ends. These sticky ends can hydrogen-bond with DNA cut by the same enzyme and be joined by DNA ligase.
Example chart: restriction enzyme EcoRI; substrate DNA containing 5'-GAATTC-3'/3'-CTTAAG-5'; cut between G and A on both strands; product has sticky ends with AATT overhangs.
DNA is a very long polymer; for example, human diploid DNA contains about 6.6 x 10^9 base pairs and even bacterial DNA contains millions of base pairs. Enzymes are proteins made of amino-acid chains and are much smaller than such chromosomal DNA molecules. The chapter also notes the need to break long DNA into fragments for sequencing and cloning.
DNA is generally bigger in molecular size than enzymes.
A diploid human cell has about 6.6 x 10^9 base pairs. Amount in moles of base pairs = (6.6 x 10^9) / (6.022 x 10^23) = about 1.1 x 10^-14 mol. Molar concentration means moles per litre, so a definite concentration cannot be calculated unless the relevant volume is specified.
In a diploid human cell, the amount of DNA is about 1.1 x 10^-14 mol of base pairs; an actual molar concentration also needs the nuclear or cell volume.
Restriction enzymes were isolated from bacteria and are part of restriction-modification systems that restrict bacteriophage growth. Eukaryotic cells do not normally use restriction endonucleases as a defence system like bacteria; their DNA is organised and protected differently.
No, restriction endonucleases are known mainly from prokaryotes, where they protect cells by cutting invading foreign DNA.
Unlike shake flasks, bioreactors can process 100-1000 litres of culture. They have agitator systems, oxygen delivery systems, foam control, temperature and pH control and sampling ports. These conditions maintain cells in active growth and improve production of recombinant proteins or other products.
Stirred tank bioreactors allow large-scale culture, control of temperature, pH, oxygen supply and foam, sterile operation, sampling, and continuous culture with higher yield.
A DNA palindrome reads the same on the two strands when both are read in the same 5' to 3' direction. Examples are EcoRI: 5'-GAATTC-3', HindIII: 5'-AAGCTT-3', BamHI: 5'-GGATCC-3', PstI: 5'-CTGCAG-3' and SalI: 5'-GTCGAC-3'.
Examples of palindromic DNA sequences include GAATTC, AAGCTT, GGATCC, CTGCAG and GTCGAC.
During prophase I, homologous chromosomes pair and exchange segments at chiasmata. This crossing over creates new combinations of alleles on chromatids, producing recombinant DNA.
In meiosis, recombinant DNA is made during pachytene of prophase I, when crossing over occurs between non-sister chromatids of homologous chromosomes.
NCERT gives beta-galactosidase as an example. If foreign DNA is inserted within the coding sequence for beta-galactosidase, the gene is inactivated. In the presence of a chromogenic substrate, non-recombinant colonies become blue, while recombinant colonies with the insert do not produce colour. Thus the reporter helps monitor successful insertion.
A reporter enzyme can reveal transformation by producing a visible colour or signal; insertion of foreign DNA can inactivate the reporter gene so recombinant colonies differ in colour from non-recombinants.
The ori sequence enables linked DNA to replicate in host cells and also influences copy number. Bioreactors maintain conditions such as temperature, pH, oxygen, mixing and nutrients so cells can produce the desired product at scale. Downstream processing begins after biosynthesis and prepares the product for marketing through purification, preservatives, clinical testing and quality control.
(a) Origin of replication is the DNA sequence where replication starts. (b) Bioreactors are vessels that provide optimal conditions for large-scale biological production. (c) Downstream processing includes separation, purification, formulation and quality testing of a biotechnological product.
PCR repeats denaturation, primer annealing and extension to make many copies of a gene of interest. Restriction endonucleases recognise palindromic DNA sequences and cut within DNA, often generating sticky ends useful in recombinant DNA construction. Chitinase digests chitin in fungal cell walls, helping release DNA during isolation of genetic material.
(a) PCR is in vitro amplification of a DNA segment using primers and thermostable DNA polymerase. (b) Restriction enzymes cut DNA at specific recognition sequences. (c) Chitinase is an enzyme used to break fungal cell walls during DNA isolation.
Plasmids act as cloning vectors because they replicate independently and may carry selectable markers, while chromosomal DNA carries most hereditary information. RNA has a 2'-OH group and is more reactive, whereas DNA is more stable. Restriction enzymes used in genetic engineering are endonucleases because they cut within DNA at specific recognition sites.
(a) Plasmid DNA is small, circular, extra-chromosomal and independently replicating; chromosomal DNA is the main genomic DNA. (b) RNA usually has ribose and uracil and is often single-stranded; DNA has deoxyribose and thymine and is usually double-stranded. (c) Exonucleases remove nucleotides from DNA ends; endonucleases cut at internal positions.