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Genetic technology

The document outlines the processes and tools involved in genetic engineering, including gene transfer, the use of vectors, and the synthesis of recombinant proteins like human insulin. It details methods such as PCR for DNA amplification and the role of bioinformatics in analyzing genetic data. Additionally, it discusses applications in medicine, such as gene therapy and the production of therapeutic proteins for genetic disorders.

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0% found this document useful (0 votes)

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Genetic technology

Uploaded by

manya.pandey

We take content rights seriously. If you suspect this is your content, claim it here.

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Download as PDF, TXT or read online on Scribd

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● Genetic engineering: modifying an organism’s genome by altering the DNA base

sequence or introducing a new gene (for it to be expressed).

● Recombinant DNA: DNA that has been modified by joining genes from one or more
species.
● GMO: an organism that has been genetically modified to produce a change that natural
breeding or artificial breeding wouldn't otherwise produce.

Process of gene transfer:

1) The desired gene is identified and either cut from a chromosome, made using mRNA by
reverse transcription, or artificially made by sequencing nucleotides.
2) The desired gene is made into many copies using PCR
3) A vector is used to deliver the gene to the cells
4) The cells that accepted that gene are identified and cloned to produce multiple copies

Where do you get the gene?

● Cut from another chromosome
● Reverse transcription from mRNA
● Synthesised using nucleotides

Tools for genetic technologists

1) Restriction enzymes

● Restriction endonucleases: Enzymes originally found in bacteria that break the
DNA of viruses that infect them, and can cut the DNA at specific locations
(location depends on the type of RE).
● Called restriction enzymes because they restrict viral infection
● Bind to specific parts of the DNA and create double-strand breaks
● Breaks are either straight through or lateral, which produces ‘sticky ends’
● Sticky ends are short lengths of single-stranded DNA which have unpaired
nucleotides that can form H-bonds with their complementary base pairs.
● Sticky ends are useful for PCR and microarray
2) Reverse transcriptase
● Found in RNA viruses
● Can synthesise DNA back from mRNA
● Useful to get the DNA without the presence of a mixture of introns and exons

3) DNA synthesis

● Computers are used to create DNA sequences
● This is used to engineer short fragments of DNA, which are later joined together

4) Vectors
● An intermediate used to transfer genes into the desired location

How to obtain vectors? (plasmids)

● One type of vector is a plasmid found in bacteria

● Bacteria are treated with enzymes to break down their cell walls, then spun in a
centrifuge to separate plasmids
● The desired gene is cut from DNA with restriction endonucleases
● The plasmid is also cut open with the same restriction enzyme to produce
complementary sticky ends
● The gene is introduced to it and fuses with the sticky ends.

How are vectors introduced into the bacteria?

● Bacteria + vector placed in a solution of high calcium ion conc.
● The solution is cooled then heat shocked to create transformed bacteria.

Synthesis of recombinant human insulin:

● Reverse transcription is used to make cDNA strands from the mRNA from B-cells in the
human pancreas.
● The DNA made is inserted into bacteria plasmids and then into bacteria through them.
● The transformed bacteria are identified and cultured and are grown in large fermenters.
● The bacteria now produce human insulin.

Advantages of Recombinant proteins:

● Different types of insulin can be manufactured depending on demand (slow-acting/fast

etc…)
● Insulin can be produced on a large scale to meet demand.
● Small amounts of space are needed to manufacture
● Cost-effective compared to animal insulin
● Bacteria can be cultured to large numbers easily requiring minimal nutrients
● Need a small area to manufacture large volumes at once
Why are promoters inserted with the new gene?

● They indicate to the RNA polymerase which strands are the template strands that need
to be transcribed.
● They allow the RNA polymerase to bind to the template strand.
● They ensure that the gene is expressed at high levels.

Marker genes?
● Genes that code for a feature that can be easily identified.
● They have the same promoter as the new gene inserted to ensure they are transcribed.
● Eg: GFP from jellyfish fluoresces under UV
● B-glucuronidase gives colour to the subject to study cell gene expression activity.

Polymerase chain Reaction:

→ It is the method used to amplify small amounts of DNA

PCR soup: Deoxynucleotides, single-stranded DNA as primers, pH 7-8 buffer solution, Heat
stable Polymerase, the DNA to be amplified

Process:
1) Denaturation: the solution is heated to 95 Celsius which breaks the H-bonds and
separates the DNA strands.
2) Annealing: The temp cools to 60 Celcius and the primers (20bp long) bind to the single
strands to allow the DNA polymerase to start making a complementary strand.
3) Extension: The mixture is heated to 72 Celcius and the DNA polymerase makes the
cDNA.
Why taq polymerase?
● Heat stable
● So it does not need to be replaced after every cycle of PCR, making it cost-effective and
efficient overall.

Bioinformatics databases:
● provide information about DNA base sequences of organism’s genomes
● Information about amino acid sequences in proteins
● Structure and folding of proteins information

Advantages of bioinformatics:
● Can store huge amounts of biodata
● Can be used to search for data and share it with the world
● Can be used to manipulate, analyse and compare different data such as protein
structures
● Can be used to study evolutionary relationships between organisms
● Visualising protein structures and genes
● Development of gene-targeting drugs such as for malaria