Describe the use of recombinant DNA technology in the synthesis of human insulin by bacteria [8] #
- mRNA coding for insulin/isolate gene for human insulin;
- from beta cells of islets of Langerhans/pancreas;
- reference to reverse transcriptase;
- to cDNA;
- reference PCR/DNA polymerase/double strand;
- reference sticky ends;
- use of vector/virus/plasmid;
- reference endonuclease/restriction enzymes;
- to cut plasmid;
- reference DNA ligase to join DNA;
- inserted into suitable host cell/E.coli/bacteria;
- reference method of insertion;
- identification of modified bacteria;
- reference growth/culture of engineered bacteria in fermenters
Describe how the gene coding for human insulin can be obtained and inserted into a plasmid vector. [8] #
- obtain mRNA from β cells (of islets of Langerhans of pancreas) ;
- reverse transcriptase ;
- make (single-stranded) cDNA ;
- DNA polymerase used to make cDNA double stranded ;
- sticky ends created ;
- (obtain) plasmids ;
- cut with restriction, endonuclease/ enzyme ; A named e.g. EcoR1
- ref. complementary sticky ends ;
- cDNA / insulin gene, mixed with plasmid ;
- DNA ligase ;
- seals nicks in sugar-phosphate backbone ;
Explain the advantages of treating diabetic people with human insulin produced by gene technology.[6] #
- it is identical to human insulin ;
- (more) rapid response ;
- no/ fewer, immune response/ side effects/allergic reactions ;
- ref. to ethical/ moral/ religious, issues ;
- cheaper to produce in large volume/ unlimited availability ;
- less risk of, transmitting disease/infection ;
- good for people who have developed tolerance to animal insulin ;
Explain how bacteria can be genetically modified and then identified using antibiotic resistance genes.[8] #
- (recombinant) plasmids mixed with bacteria ;
- (some) bacteria, take up plasmids/ transformed ;
- heat shock / calcium chloride solution/Ca 2+ ions/electroporation ;
- to identify bacteria containing plasmids
- grow on, agar/medium, containing antibiotic (A) ;
- plasmid contains, antibiotic (A)/ ampicillin, resistance gene(s) ;
- bacteria with plasmid survive ;
- to identify recombinant bacteria
- replica plate ;
- (onto) agar/ medium, containing second antibiotic (B) ; A tetracycline
- resistance gene inactivated (by insertion of new, DNA/ gene
Outline how genetic fingerprinting is carried out [8] #
Describe how electrophoresis is used in genetic fingerprinting [8]
- ref. to VNTR (sequences) ;
- quantity of DNA increased by PCR ;
- DNA fragmented by, restriction enzyme(s) / endonuclease(s) ;
- loaded (into wells) in agarose gel ;
- (at) negative end/ cathode end ;
- ref. to buffer/ electrolyte ;
- direct current applied ;
- phosphate groups of DNA give negative charge ;
- (negatively charged) DNA attracted to, anode/ positive electrode ;
- short pieces/ smaller mass, move further/move faster ;
- (pieces) transferred to, membrane/ nylon/nitrocellulose/ absorbent paper or Southern blotting ;
- heated to separate strands ;
- probes/ fluorescent dye, added ;
- X-ray film/ UV light/ lasers ;
- pattern of stripes/ ref. banding pattern ;
Decsribe how genetic screening is carried out in an adult human [8] #
- samples of cells taken
- e.g white blood cells
- test for chromosomal abnormalities
- e.g Down’s Syndrom
- cells cultured so that they divide
- stopped at metaphase
- use of colchicines
- cells made to take up water
- chromosomes separated
- karyotype produced
- flourescent makers to help detect abnormalities
- test for particular allele
- DNA extracted
- DNA cut with restriction enzyme
- electrophoresis occurs
- to separate DNA into strands
- complementary/specific probe(for mutant allele)
Discuss the advantages of screening for genetic conditions[8] #
- information about the increased risk of person having genetic conditions ;
- ref. breast cancer/ named example ;
- allows people to prepare for late onset genetic conditions ;
- ref. Huntington’s disease/Alzheimer’s disease/named example ;
- identify whether fetuses are going to develop a genetic condition ;
- so can give early treatment when born ;
- allows parents to prepare for the birth of a child who will need treatment for a considerable time or even throughout life ;
- identifies carriers of genetic conditions ;
- helps to provide early diagnosis ;
- allows couples who are both carriers of a genetic condition to make decisions about starting a family/ having more children/ seeking IVF ;
Describe the ways by which gene mutations can occur. [6] #
- change in, base / nucleotide, sequence (in DNA) ;
- during DNA replication ;
- detail of change ; e.g. base, substitution / addition / deletion
- frame shifts /;
- different / new, allele ;
- random / spontaneous ;
- mutagens ;
- ionising radiation ;
- UV radiation / mustard gas ;
Explain how genetic diseases may be treated using gene therapy. [6] #
- normal, gene/ allele ;
- (insert into) vector ;
- liposomes (as vectors) ;
- liposomes in, aerosol/ inhaler ;
- liposome fuses with host cell ;
- virus (as vector) ;
- virus vector harmless ;
- short term effect ;
- repeat treatments needed ;
- side effects ;
Explain how changes in the nucleotide sequence of DNA may affect the amino acid sequence in a protein. [7] #
- code is three, bases / nucleotides ; triplet code
- (gene) mutation ; chromosome mutation
- base, substitution / addition / deletion ;
- addition / deletion, large effect (on amino acid sequence) ;
- frame shift ;
- completely new code after mutation / alters every 3 base sequence which follows ;
- (substitution) often has no effect / silent mutation ;
- different triplet but same amino acid / new amino acid in non-functional part of protein ;
- (substitution) may have big effect (on amino acid sequence) ;
- could produce ‘stop’ codon ;
- sickle cell anaemia / PKU / cystic fibrosis ;
- reference to transcription or translation in correct context ;
Explain the advantages and disadvantages of genetic screening in humans. #
- Advantges
- known risk for next generation;
- allows possible abortion of affected fetus;
- allows sorting of IVF embryos;
- Disadvantages
- loss of hope;
- insurance company problems;
- employment problems;
- miscarriage risk;
- trauma of knowledge;
