Recombinant DNA technology, and use of genes

What trait of DNA does recombinant DNA technology rely on (2)

genetic code is universal, each mRNA codon codes for the same amino acid in all organisms. Transcription and translation are also universal

5 stages involved in recombinant gene technology

1. acquire the target gene

2. amplify (replicate) the gene

3. insert gene into vector

4. insert vector into host organism

5. identify the genetically modified organism

1. 3 methods to acquire the target gene

1. use mRNA and reverse transcriptase

2. using restriction endonucleases

3. using a gene machine

explain acquiring the target gene by using mRNA and reverse transcriptase (4)

1. obtain mRNA from cytoplasm of cell

2. Add DNA nucleotides and reverse transcriptase

3. produce single-stranded cDNA that only contains exons

4. therefore can be expressed by prokaryotic organisms

using restriction endonucleases to acquire target gene

1. enzyme that cuts DNA

2. at specific base sequences, called recognition / restriction

3. forming sticky ends - short lengths of single stranded DNA

4. that can form hydrogen bonds with complementary sticky ends

using a gene machine to acquire target gene (2)

1. type the required base sequence into a computer

2. machine assembles the nucleotides together to form the gene

amplifying the gene aka…

dna replication in vitro

what reaction does amplifying the gene use

polymerase chain reaction (PCR)

requirements for amplifying the gene (4)

1. target / desired gene to act as a template

2. dna nucleotides

3. (thermostable / taq) dna polymerase

4. dna primers

dna primers (2) and function (2)

• short length of single strand DNA

• complimentary base sequence to the start of the DNA

• allows for DNA polymerase

• prevents DNA strands rejoining

2. Describe how to amplify the gene in vitro

uses polymerase chain reaction (PCR

1. Heat the target DNA to 95 degrees to break hydrogen bonds and separate the strands

2. Reduce temperature to 55 degrees to allow complementary primers to bind to DNA

3. Increase temperature to 72 degrees to allow the DNA polymerase to join the nucleotides together. forming phosphodiester bonds

4. Repeat the cycle

Plasmid (3)

• small circular DNA molecule

• contain additional genes eg. antibiotic resistance

• only found in some bacteria

3. Steps involved in inserting gene into a vector in vivo

1. cut the plasmid DNA with the same restriction endonuclease used to isolate target gene, producing sticky ends - hydrogen bonds form between complimentary sticky ends

2. DNA ligase joins the target gene to the plasmid, catalysing the formation of phosphodiester bonds

3. produces a molecule of Recombinant DNA, containing genes from two different organisms

Enzymes involved in inserting gene into vector in vivo (2)

1. same restriction endonuclease from acquiring the target gene

2. DNA ligase to form phosphodiester bonds between target DNA and plasmid

4. how to insert genes into a host cell (2)

plasmids are added to culture of growing bacteria

some bacteria will take up plasmid

5. Identifying GM bacteria (7)

• uses plasmid with marker genes eg. genes for antibiotic resistance eg antibiotic A resistance gene. target gene is inserted into marker gene, so marker gene no longer expressed

• at the end of stage 4, there will be: bacteria with no plasmids, normal plasmids, and recombinant plasmids

• bacteria is grown on different agar plates

• master plate: no antibiotics, all bacteria form colonies

• antibiotic b plate: contains antibiotic B, bacteria without plasmids will not form colonies

• antibiotic A plate: contains antibiotic A, only bacteria with normal plasmid will form colonies, GM bacteria die

• GM bacteria identified by being on master and antibiotic B plate, but not antibiotic A

6. Process of growing GM bacteria to produce human protein (3)

1. GM bacteria are removed from master plate and cultured

2. as bacteria replicate by binary fission, human gene is replicated at each generation

3. bacteria express gene, human protein is produced

Gene therapy use and what it involves (3)

• application of recombinant DNA technology

• used to treat genetic conditions

• involves introduction of normal allele to replace / supplement mutant allele

vectors used in gene therapy (2)

• liposomes

• virus

liposomes definition (1) and mechanism (2)

• small spheres of phospholipids containing recombinant dna

• fuse with cell surface membrane of target cell

• dna released into cytoplasm and expressed in the nucleus

virus use in gene therapy (2)

• attenuated so that it is not a pathogen

• attaches to cell surface membrane, DNA enters cell and enters nucleus

pros and cons of virus and liposome in gene therapy

liposome

• pro: cannot mutate to become pathogenic

• con: may not enter nucleus and therefore may not be expressed

virus

• pro: will enter nucleus, so more likely to be expressed

• con: may mutate and become pathogen

DNA profile / genetic fingerprint (3)

• introns / non-coding regions of DNA contain variable number tandem repeats (VNTRs) aka short tandem repeats (STRs)

• unique for every individual

• based on genes and epigenetics

Steps to create a genetic fingerprint (5)

1. obtain individual’s DNA

2. amplify sample using PCR

3. use restriction endonuclease to cut DNA into fragments. a mixture of different lengths of fragments is formed

4. use gel electrophorisis to separate the fragments

5. use gene probes to identify fragments

explain gel electrophorisis (4)

• DNA mixture is loaded into well at one end of agarose gel

• agarose gel is covered in a saline solution and a direct current applied

• negatively charged DNA molecules move toward positive electrode

• shorter fragments are lighter, so move faster, so move further

why are DNA and proteins negatively charged

DNA: phosphate group

proteins: R groups on amino acids

explain the dna marker / ladder (2)

• mixture of DNA fragments of known length

• can be used to estimate length of unknown DNA fragments

explain using gene probes to identify fragments (6)

• probe is radioactively labelled

• dna fragments are transferred to a nylon mesh and treated to produce single stranded DNA

• labelled probes are added to the mesh and will bind to DNA by DNA hybridisation

• photographic film is placed onto hybrid mesh

• radioactive probe causes film to “fog”

• by comparing banding patterns, relationships can be established

dna fingerprints can be used in (4)

1. forensic analysis

2. paternity testing

3. phylogenetics + genetic variation within species, including sub-populations

4. medical screening / diagnosis of inherited / genetic conditions

how is gene fingerprinting used in medical screening

• genetic probes are created to have a similar base sequence to the mutated allele / oncogene