Chapter 21 - Recombinant DNA Technology Flashcards
What is recombinant DNA technology?
The process by which genes can be manipulated, altered and transferred from one organism to another
State the importance of DNA technology
A lot of human disorders are caused by an individual being unable to produce a particular protein themself
- DNA technology allows cloning of a gene of interest to produce large quantities of this gene and desired protein, which can be given to help human disease
What does ‘to clone’ mean?
To make identical copies of
What are the two ways cloning of a gene can occur?
- In Vivo = within a living organism - the gene is transferred into a host cell to be cloned
- in Vitro = outside a living organism - in a lab, the gene is copied using a method called PCR
Outline the 5 key steps involved in in vivo cloning of a gene
- Isolates the gene of interest from healthy human cell
- Insertion of this gene of interest into a vector (usually a plasmid)
- Transformation = inserting the carrier molecule into a bacterium cell
- Identification of those bacteria that contain the vector
- Growth of bacteria via binary fission.
What does the term recombinant DNA mean?
Taking DNA from 2 sources and combining
IN VIVO STEP 1 - Isolation of the desired gene
What are the 3 ways this can be done?
- Using reverse transcriptase to convert mRNA of the gene of interest into DNA
- Using restriction endonuclease to cut out the fragment of DNA containing the desired gene of interest from the DNA
- Creating the gene in a gene machine
IN VIVO STEP 1 - Isolation of the desired gene
Explain how using reverse transcriptase works to isolate gene of interest
- B cells from islets of langerhans in humans pancreas are specialised to produce insulin + make a lot of mRNA for insulin
- mRNA acts as a template in which a single stranded complementary copy of DNA (cDNA) is formed using reverse transcriptase
- cDNA is isolated by hydrolysis of mRNA with an enzyme
- Double stranded DNA is formed on template of cDNA using DNA polymerase = copy of human insulin gene
IN VIVO STEP 1 - Isolation of the desired gene
Explain how using restriction endonuclease works to isolate gene of interest
Restriction endonuclease:
- enzymes that cut double stranded DNA
- by breaking phosphodiester bonds in backbone
- each enzyme recognise and cuts DNA at a specific target site (specific sequences of bases) called restriction sequences (4-6 bases long)
- cuts either creates blunt ends = line of cut is straight
- or sticky ends = line of cut is staggered
IN VIVO STEP 1 - Isolation of the desired gene
How does creating the gene in a gene machine work?
(No introns as working back from protein = easier)
The desired protein is used to work out the DNA sequence of the gene coding for that protein
- The amino acid sequence of protein is determined
- From this the mRNA codons are looked up using a conversion table
- From this complementary DNA triplets are worked out
- the DNA base sequence for the gene is then fed into a computer which designs and assembles the gene (bioformatics)
IN VIVO STEP 2 - insertion of gene into a vector
Outline what occurs
(Assumes restriction endonuclease was used for isolating gene of interest)
Sticky ends are useful if 2 sources are cut with same restriction endonuclease enzyme = complementary
- hybridisation = forming of hydrogen bonds between 2 sources of DNA that have been cut = now recombinant DNA
- DNA ligase forms phosphodiester bonds between 2 sources of DNA
IN VIVO STEP 3 - transformation into host cell
Explain the possible outcomes
The more permeable the bacterial cell membrane, the more substances can move in + out (vector) = affected by temp + calcium ions
- 99% non transferred - only some bacterial cells will take up the vector when mixed
- 0.1% successful transformation - but vector does not contain gene of interest
- Very few - successfully transformed and transgenic (contains gene of interest)
IN VIVO STEP 4 - identification
How is it done?
Using antibiotic resistance genes to find these bacteria that do have the gene of interest
- insertional inactivation of marker gene
Two plates (one containing ampicillin bacteria and other containing tetracycline)
- transfer is done by pressing a nylon sheet gently into surface of ampicillin plate + then into tetracycline plate
- then find those that die on 2nd plate + go back to other
IN VIVO STEP 5 - growth/ cloning of selected bacteria
Outline how this is done
Select the identified transgenic bacteria and grow them (allow to reproduce) so they produce lots of the desired gene + protein
Other than antibiotic resistant marker genes, outline two others
Fluorescent marker genes
Enzyme marker genes
Outline how IN VITRO cloning is done?
PCR = polymerase chain reaction
Amplifying a single copy of a DNA segment of interest into billions of identical copies
What are the ingredients for a thermocyler?
Thermocycler = computer controlled machine that varies the temp over time
- Taq DNA polymerase
- buffer
- DNA
- DNA primers
- free DNA nucleotides
Outline role of Taq DNA polymerase and DNA primers in IN VITRO cloning
Taq DNA polymerase = thermally stable so can tolerate heat and adds free nucleotides to strands
DNA primers = short single stranded sequences of nucleotides that are complementary to end of target DNA segment
Explain the process of IN VITRO cloning using a thermocycler
- Hydrogen bonds between 2 DNA strands break due to high temperature (95˚c) = separates strands
- Then cooled to around 55-60˚c which causes DNA primers to anneal to complementary sequences at ends of DNA fragments
= a starting point for Taq polymerase - Temperature is then raised to 72˚c which is optimal for Taq polymerase to work at - it adds free nucleotides to primers along each of strands
What does the term anneal mean?
Forming hydrogen bonds
What are DNA probes?
a short, single-stranded length of DNA that has some sort of label attached to it that makes it easily identifiable to us by eye.
- It identifies a specific target sequence because it is complementary to the gene or part of the gene of interest
What are the 2 commons ways of labelling using DNA probes?
- Radioactively labelled probes - made of nucleotides containing P32 (phosphorus). The probe is then identified using an x-ray film exposed by radioactivity
- Fluorescently labelled probes - emit light (fluoresce) under certain conditions (eg. when it successfully binds to the particular target sequence) which can be viewed under UV light.
Outline the process of using DNA probes to locate a gene [5]
● DNA probe is made so that it has a base sequence that is complementary to part of the gene of interest
● The double stranded DNA that contains the gene of interest is heated to separate the two strands
● The separated DNA strands are mixed with the probe + cooled
● The probe can then bind to its complementary sequence on one of the strands by hydrogen bonding (annealing/hybridisation)
● The site at which the probe binds can then be identified by radioactivity or fluorescence emitted by the probe
What are the uses of DNA probes in human genetic disorders?
- Probes may be useful to determine whether someone possesses a mutant allele of a gene (that causes a particular genetic disorder/disease) - so individuals can be screened for a mutant allele that may cause a particular disease
- Many genetic disorders are the result of gene mutations
What does the term genetic library mean?
Stores the base sequences for specific alleles/ genes
- usually used to determine base sequences of mutant alleles that cause diseases
What does screening for genetic disorders refer to?
Important that individuals with a family history of genetic disorders to be screened as they may carry mutant alleles
- can determine the probability of a couple having offspring with the genetic disorder (2 recessive alleles coming together or inheriting 1 allele but developing another via mutation)
What’s the advantage of genetic screening?
+ ability to use personalised medicine based on genotype
What is genetic fingerprinting?
The analysis of DNA samples from samples of body tissues or fluids, usually used to identify individuals (personal identification)
What are the 5 key stages of genetic fingerprinting?
- Extraction
- Digestion
- Separation of DNA fragments
- Hybridisation
- Development
Stage 1 Genetic Fingerprinting
Outline the process of extraction
DNA is extracted from an individuals sample by separating the DNA from the rest of the cell
- As the amount of DNA in a sample is usually quite small, it can be increased using PCR
Stage 2 Genetic Fingerprinting
Outline the process of digestion
As stage 3 requires DNA fragments, restriction endonuclease enzymes are used to cut out the DNA from the sample into fragments
Stage 3 Genetic Fingerprinting
Outline the process of separation of DNA fragments
- Gel electrophoresis is a method used to separate DNA fragments by size, and then analyse DNA fragment bands
- The gel is immersed in alkali in order to separate the double strands into single strands
- DNA fragments are transferred from gel to nylon membrane
Stage 4 Genetic Fingerprinting
Outline the process of hybridisation
- probes can be used to label the fragments to be visualised (radioactively/ fluorescently labelled)
- Probes have a sequence that is complementary to sequences of VNTR’s + and bind under certain conditions (pH + temp)
- Then washed to remove any remaining DNA probe
Stage 5 Genetic Fingerprinting
Outline the process of development
The probes position in the gel can be determined (using UV light/ x-ray)
What are VNTR’s?
Variable Number Tandem Repeats
- repetitive non-coding regions of DNA
- for every individual, the sequence is the same but number of repeats of VNTR’s is unique
- so VNTR’s can be used as markers for personal identification
How are the results of genetic fingerprinting interpreted?
The pattern of bands is unique to every individual
- DNA fingerprints from 2 DNA samples can be compared for similarity
- the closer the match between patterns, the greater the probability the 2 samples come from same person/ closely related
What are the 4 key uses of genetic fingerprinting?
- Resolving genetic relationships
- Forensic science
- Medical diagnosis
- Plant and animal breeding
Outline how genetic fingerprinting can be used to resolve genetic relationships
A child inherits half of genetic material from mother and half from father
- so VNTR’s will be similar to that of parents
Outline how genetic fingerprinting can be used in forensic science
DNA is often left at scene of crime
- DNA fingerprinting can identify whose it is to see if they were there at the time of the crime (not who did the crime necessarily)
Outline how genetic fingerprinting can be used in medical diagnosis
Genetic fingerprints can help in diagnosing diseases
● A sample of DNA from a person with the mutant allele for a disease can be cut using restriction endonucleases and a DNA fingerprint prepared
● This can then be compared to the fingerprint of other individuals to see if they have the mutant allele
Outline how genetic fingerprinting can be used in plant + animal breeding
To prevent undesirable inbreeding on farms or in zoos - mate animals whose fingerprints differ the most (so two recessive alleles don’t come together)
● Can also be used to identify plants or animals that have a desirable gene (for GM) = selected for breeding
