Flashcards in 14. DNA Manipulation Deck (63):
What does DNA manipulation refer to?
Altering an organism's DNA by either adding new DNA or editing existing DNA
Genetic engineering refers to...?
The scientific method for the artificial manipulation of genes
What is a restriction enzyme?
-Endonuclease is a tool used by genetic engineers to cut DNA
-cuts DNA at precise sequences of 4-8 base pairs called recognition sequences. Once recognised the enzyme binds to DNA and cuts it in a fixed and predictable way
Where do restriction enzymes come from
Occur naturally in Bacteria, thought to have evolved as a defence mechanism against viruses
What is a recognition sequence?
The position where a cutting enzyme can snip its recognition sequence and is where a particular order of nucleotides occurs
Difference between blunt and sticky ends?
-Some restriction enzymes cut the two strands of a DNA molecule at points directly opposite to each other to produce blunt ends. Can be joined to other sticky ends with a complementary base sequence
-Other cutting enzymes cut one strand at a point but the second strand at a point that is not directly opposite. Known as sticky ends which are complementary
What is CRISPR
A complex comprising cas9 Endonuclease and sgRNA.
Cuts DNA at very specific sequences and can be used to edit genes
Where is CRISPR cas9 usually found
Occurs naturally in bacteria, which use it to chop the DNA of invading viruses
What two components are required for CRISPR to work
An RNA guide, sgRNA that locates and binds to the target piece of DNA and the cas9 Endonuclease that unwinds and cuts the DNA
What potential does the CRISPR cas9 technology have?
This technology has the potential to correct mutations responsible for genetic diseases, switch faulty genes off add new genes to an organism or to study the effects of specific genes
Guided to the target site by sgRNA. Cas9 unwinds the DNA and cuts both strands at a specific point
Single guide RNA
Is a shot synthetic RNA sequence designed to guide cas9 to the site of interest (eg a faulty gene sequence). It contains a nucleotide section which is complementary to the DNA of interest
What is the pam sequence
It lies directly downstream of the target sequence on the non target DNA strand. Recognition of PAM by cas9 destabilises the DNA allowing the sgRNA to be inserted. Cas9 will not function if PAM is absent
Gene knock in (gene editing)
A new DNA sequence is inserted into the DNA break. For example allows a faulty gene sequence to be replaced with the correct sequence to restore normal gene function
Gene knock out (gene silencing)
As the cells normal repair process mend the broken DNA errors occur resulting in the insertion or deletion of a nucleotide bases. The resulting frame shift mutation changes the way the nucleotid sequence is read, either disabling gene function or producing a STOP signal. Can silence faulty genes
Why does DNA move in electrophoresis?
-DNA is negatively charged, due to their phosphate group in their sugar phosphate backbone
-DNA moves from negative terminal to positive terminal because DNA is negatively charged and is attracted to the positive charge at the positive terminal
What determines the speed at which fragments move?
The shortest DNA fragments move most quickly and are found further away from the starting point. The longest fragments move most slowly and are found closer to the starting point. Fragments of the same size move at the same rate.
What are standards
Consist of DNA fragments of known length and are used to compare the size of the sample DNA
How are DNA fragments made visible after the gel run?
The separated DNA bands must be made visible either through the use of a due or a radioactive probe.
One technique makes use of ethidium bromide which binds to major groove of DNA molecules.
When illuminated by u,tra violet light the DNA bound to etbr fluorescent pale pink
What is a probe?
-A probe is a single strand of DNA or RNA with base sequence that is complementary to the base sequence in one of the strands of the target DNA.
-Labelled with a radioactive or fluorescent marker so that the location of the probe and hence the target DNA can be seen.
Types of tag
Fluorescent dye tag: shows up as fluorescent bands when gel is exposed to ultraviolet light source
Radioactive tag: shows up as a dark band when the gel is exposed to photographic film
What might gene probes be used to search for?
-The position of a gene on a chromosome
-The presence of an allele of a specific gene associated with a genetic disease
-The genetic finger print of a person to identify them (paternity testing, forensic identification of subjects).
Southern blotting process
1. The DNA of interest is extracted from living or dead organism, or DNA found at crime scene
2. It is cut using restriction enzymes, leaving thousands of fragments of different sizes
3. DNA is separated using gel electrophoresis
4. DNA fragments transferred by blotting to membrane. DNA is made single stranded and fixed in place on the membrane.
5. Filter sheet immersed in a bath with radioactive probes. When a probe finds a complementary base sequence it will join to the other strand. Hybridisation. Pairing between single stranded complementary DNA segments
6. The probe, and hence the DNA of interest, is located, either by use of X-ray film in the case of a radioactively labelled probe where the radioactive probes show up as dark bands on X-ray film, or by fluorescence in the case of a probe with a fluorescent label.
What is ligation
The process by which DNA fragments using restriction enzymes can be joined.
What happens in the process of ligation
-An enzyme, DNA ligase catalyses the joint of pieces of double stranded DNA at the sugar phosphate backbone.
-joining can produce one larger piece of DNA or a circular molecule of DNA
-two pieces of DNA of different origins (two different species) can be joined together using restriction enzymes and DNA ligase to produce recombinant DNA
How are plasmids used in gene insertion
Vectors can transport DNA fragments (passenger DNA) into cells.
Vectors are found in bacterial cells. Small circular pieces of DNA which are able to replicate independently of the bacterial cells chromosome
Large copies found.
Carry antibiotic resistance marker genes
Define recombinant plasmid
A plasmid that contains DNA from two or more different sources
Steps in ligation
1. Two pieces of DNA are cut using the same restriction enzyme producing same sticky ends (eg plasmid and foreign gene)
2. When the two matching sticky ends come together they join by complementary base pairing, annealing. These fragments are attracted to each other by weak hydrogen bonds. Annealing stabilises the molecule so that they can be permanently joined
3. The fragments of DNA are permanently joined together by DNA ligase, producing a recombinant DNA. DNA ligase catalyses phosphodiester bond between sugar and phosphate group in the DNA backbone
Name two ways of making DNA fragments
1. Synthesis DNA from nucleotide building blocks.
2. Make a copy of DNA using an mRNA template
What is the process of synthesising DNA from nucleotide building blocks
-uses DNA synthesiser
-the base sequence of the required DNA must be known
-DNA synthesisers join nucleotide sub units in a predefined order to produce DNA segments with lengths greater than 100 bases
-chemical synthesis of DNA does not require a template strand or enzyme DNA polymerase
Primers or probes
How is a copy of DNA made using an RNA template
1. Double stranded DNA of a gene from a eukaryotic organism contains introns
2. As a normal part of the cell process of gene expression, transcription creates a primary RNA molecule
3. The introns are removed by restriction enzymes to form mature mRNA (now excluding the introns) that codes for the making of a single protein
4. mRNA extracted from the cell and purified
5. Reverse transcriptase is added which synthesises a single stranded DNA molecule complementary to the mRNA
6. The second DNA strand is made by using the first as a template and adding the enzyme DNA polymerase
Why are introns removed when making a DNA copy via mRNA template?
-in cases of in-vivo cloning it makes the DNA shorter and easier to insert into plasmids
-large amounts of non coding DNA are not made by pcr
-in case of in vivo cloning allows the bacterial enzymes to properly translate the human gene from reassembled DNA
What dos the polymerase chain reaction do?
Researchers are able to create large quantities of trace amounts of DNA. Small quantities of DNA are amplified. Uses thermal cycler
What are the sources of trace DNA?
Crime scene: single root hair, dried blood stain, drinking glass
Extinct animals: fragments of DNA from extinct animal
Medical: a single virus particle (infection), foetal cells obtained from amniotic fluid
What are the materials required for PCR?
🔸 target sequence of DNA
🔸 free nucleotides
🔸 primers (short segments of single stranded DNA)
🔸 DNA poly,erase enzyme (taq polymerase)
Steps in the process of pcr
1. DENATURATION: Heat briefly to separate DNA strands
🔸 when heated to 94° the weak hydrogen bonds between bases break and DNA dissociated into single strands
2. ANNEALING: cool to allow primers to form hydrogen bonds with ends of target sequences.
🔸 the mixture is cooled to 55°. Primers are added which pair with regions at either end of the DNA region of interest.
3. EXTENSION: DNA polymerase adds nucleotides to 3' end of each primer.
🔸 the mixture is heated to 72°. The polymerase enzyme uses the primers as a starting point and extends them so that two complete double strands are formed. For this step a supply of DNA nucleotides must be available.
Why are plasmids self replicating?
Self replicating DNA molecule because it contains a a point of origin, a base sequence where DNA replication begins
What are the properties which all vectors must have?
-be able to replicate independently inside their host organism
-have one or more sites at which a restriction enzyme can cut
–have some kind of genetic marker that allows them to be easily identified
What does transformed?
When foreign DNA is taken up from an external source by bacterial cells, those bacteria are said to be transformed.
Steps in preparation of molecular clone/ recombinant plasmid
1. A gene of interest (DNA fragment) is isolated from cells that have been grown in laboratory culture.
2. An appropriate plasmid vector is isolated from a bacterial cell
3. Both the human DNA and the plasmid are treated with the same restriction enzyme to produce identical sticky ends so they can bind.
4. The restriction enzyme cuts the plasmid DNA at its single recognition sequence, disrupting the tetracycline resistance gene.
5. The DNA fragments are mixed together and the complementary sticky ends are attracted to each other by base pairing. The enzyme DNA ligase is added to bond the sticky ends.
6. The recombinant plasmid, or molecular clone, is introduced into a bacterial cell by adding the DNA to a bacterial culture. Under right conditions some bacteria will take up the plasmid from solution by the process of transformation.
Purpose of antibiotic resistance marker genes
-used to identify the bacteria that have taken up the foreign DNA. The plasmid used often carries two genes that provide bacteria with resistance to ampicillin and tetracycline.
-a single restriction enzyme recognition sequence lies within the tetracycline resistance gene. A foreign gene spliced into this position will disturb the resistant gene leaving it vulnerable to the antibiotic.
What is electroporation?
Method in which bacterial cells are briefly placed in an electric field that shocks them and appears to create holes in their plasma membranes so that plasmid entry is facilitated.
Heat shock the bacterial cells by suspending them in an ice cold salt solution and then transferring them to 42° for less than one minute. This treatments increases fluidity of plasma membrane of the bacterial cells and increases chance of uptake of plasmids
Three possible outcomes of bacterial transformation
🔸 bacteria that haven't taken up the plasmid
🔸 bacteria that have taken up the recombinant plasmid
🔸 bacteria that have taken up the non recombinant plasmid
Foreign gene from two species
How do you identify colonies of bacteria that carry the recombinant plasmid
Differential responses to antibiotics, resistant to ampicillin but sensitive to tetracycline.
How does gene cloning occur?
🔸 the bacterium with the recombinant plasmid is allowed to multiple and express the gene
🔸 once inside the bacterial cell the plasmid and its foreign DNA (human gene), the recombinant plasmid, multiple to produce many copies.
🔸 after about 20 minutes the bacterial cell itself divides by binary fission to produce two daughter cells
🔸 bacteria continue to multiply along with the plasmid and the human DNA making millions of copies of bacterial cells and human DNA.
🔸 once bacteria carrying human gene has been isolated, human genes switched on producing protein product
How is recombinant DNA technology more effective
Replaces former means of production invoking extraction of these proteins from tissue samples from living donors or from cadavers.
Extraction from cadavers involved risk of transmission of viruses or prions
Refers to testing of individuals to detect those with the allele responsible for a particular genetic disorders
Adult screening for increased risk of disease
🔸 genetic test can be done based on family medical history. To identify if an individual has inherited a gene known to cause a particular disease. Eg BRCA1 and 2 genes that cause breast and ovarian cancers
🔸 some women who test positive for breast cancer genes make the decision to have a double mastectomy and their ovaries removed. This is a drastic step and some believe with the reliability of screening it is not necessary to take such drastic precautions
🔸 to identify people who carry a mutant allele of a gene that can be passed onto offspring. They themselves show no signs of the disease but these is a family history of the disease. Eg haemophilia
Prenatal screening and diagnosis
🔸 offered to women during their pregnancy. Tests could include blood tests ultrasound and cell free screening. Tests can indicate the genetic status of an embryo or fetus where a specific genetic disorder is suspected or it signals a birth defect.
🔸 can be invasive, slight risk of miscarriage. Some prospective parents face a difficult decision about whether to continue the pregnancy. Alerts parents and doctors for early detection and medication.
To detect gene mutations associated with disorders where symptoms appear later in life. Often requested by people with a family history of a disorder
🔹 knowledge that one has a gene mutation resulting is a disorder can have a psychological effect on the individual and family.
Also known as preimplantation genetic diagnosis is a test that involves the removal of a single cell from an embryo conceived through Ivf. The embryo is tested for genetic disorders before it is implanted into a woman's uterus.
🔸 form of DNA fingerprinting used to compare base sequence of two or more individuals.
🔸 identifies DNA from different individuals based on variable regions known as short tandem repeats or microsatellites.
🔸 detection of variation uses a combination of a single locus probe, each specific to a different Str.
Compare DNA profiling and fingerprinting
DNA profiling makes use of microsatellites (2-5 bp)
DNA fingerprinting makes use of minisatellites (9-80 bp)
What are short tandem repeats
🔸 hype variable regions of chromosomes where sequences of just two to 5 base pairs are repeated over and over- these are the microsatellites
🔸 STRs vary greatly between individuals (hypervariation) and each variation is a distinct allele
🔸 in most cases the alleles at an STR locus are named according to the number of repeats
How are STRs inherited?
🔸 at each Str locus an individual is either homozygous or heterozygous and so can have a maximum of just two different alleles
🔸 alleles inherited in Mendelian fashion, codominant. Where two different alleles of the same Str marker are present both can be detected
Source of DNA for profiling
🔸 nucleus or mitochondria.
🔸 nuclear DNA contains STRs that can identify individuals with accuracy because all individuals have unique DNA profile
🔸 mitochondrial DNA is not used to identify individual people as fa,ily members of the same maternal line have same base sequences
🔸 mt DNA used to identify victims of mass disasters or badly composed bodies where victim cannot be identified through physical means
Uses of DNA profiling
Mass disaster victim identification
Identification of human remains
Protection of Australia's wild life against unlawful collection and exportation
Examining ancient DNA
What STRs are used for forensic identification and why?
🔸 nine from different chromosomes.
🔸 chosen because they are reproducible, robust, easy to score, highly informative and have low mutation rates
🔸 tenth marker used for genetic identification
Technique used for DNA profiling
🔸 each Str loci amplified by pcr using specific fluorescent primer
🔸 products of pcr undergo electrophoresis and the fluorescent primers will emir green blue or yellow colour when exposed to uv light
🔸 a person shows one or two peaks where a peak corresponds to an allele depending on whether they are homozygous or heterozygous.
🔸 DNA profile then produced