Genetic Technologies Flashcards
define recombinant DNA technology
the transfer of DNA fragments from one organism to another
-first step is to produce/ isolate the fragments of DNA to be recombined with another piece of DNA
3 methods used to create fragments of DNA
-using reverse transcriptases to make DNA from mRNA
-use restriction enzymes to cut out a section of DNA from the genome
-use a gene machine to build a section of DNA using nucleotides.
process of using reverse transcriptase to produce DNA fragments
-a cell that naturally produces the protein made by target gene is selected
-these cells will contain lots of mRNA for the protein, which is complimentary to the target gene
-the reverse transcriptase enzyme joins the free DNA nucleotides with complementary bases to the mRNA sequence to make DNA.
-single stranded DNA is produced-> cDNA (complementary DNA as it matched the mRNA)
advantage of producing DNA fragments using reverse transcriptase
the cDNA is intron free- only contains the coding regions as it is based on the mRNA template strand
process of using restriction enzymes to produce DNA fragments
-restriction endonuleases cut DNA at specific sequences.
-naturally occur in bacteria as a defense mechanism
-each enzyme cuts DNA at a specific recognition sequence, complementary to their active site
-restriction enzymes form sticky ends (pieces of DNA that have exposed nucleotides on a single strand at each end)
-allows different fragments to be joined or annealed together, joining complementary base pairs -when cut with the same restricive endonuclease
-the restriction sequence is palindromic as they are complementary to each other
two ways DNA fragments can be amplified
-in vitro cloning- polymer chain reaction (PCR)
-in vivo cloning- using host cells
how is DNA fragment modified before inserting into a vector
-desired gene is isolated using restriction endonuclease enzymes- cut at staggered point to produce sticky ends
-promoter region and terminator regions are added to the gene to make sure the gene can correctly transcribe once in the host
->promoter: tells RNA polymerase here to start transcribing mRNA
->terminator: tells RNA polymerase when to stop
process of inserting DNA fragment into a vector
-the plasmid is cut using the same restriction endonuclease enzyme to create same sticky ends on both DNA fragment and plasmid.
-DNA fragment sticky ends have the complementary base pairs as on the sticky ends of plasmid
-DNA fragment and cut plasmid are combined together using ligase enzymes (anneals them)
-ligase enzymes catalyse condensation reactions to form phosphodiester bonds between the nucleotides.
what is a vector
something that can carry the isolated DNA fragment into the host cell e.g. plasmids, viruses and certain bacteria
process of inserting vector into host cell
-known as cell transformation
-cell membrane of host cell (usually bacteria) must be more permeable
-to increase permeability, host cells are mixed with Ca2+ and heat shocked (sudden increase in temperature)
-this enable vector to enter host cell’s cytoplasm
process of identifying transformed cells
marker genes are used to identify which bacteria succcessfully took up recombinant plasmid-they are inserted into vectors the same time the DNA is, 3 different marker genes used are:
-antibiotic resistance genes-> colonies of bacteria are grown on an agar plates that contains the antibiotic. only transformed bacteria with the resistance gene will survive + can then be selected
-genes coding for fluorescent proteins-> after they have been left to grow, a UV light can be used to identify colonies which will glow.-must be the ones with the target gene so can then be selected
-enzyme marker genes-> enzyme lactase can turn a certain substance blue from colourless, gene coding for lactase is inserted into plasmid. The DNA fragment is inserted into the middle of this gene to disrupt it, bacteria are then grown on agar plate containing colourless substance. Plasmids that cannot turn the substance blue must contain the recombinant plasmid.
reasons why host cell may not successfully take up a recombinant plasmid
-recombinant plasmid may not get inside the cell
-the plasmid re-joins to itself before DNA fragment enters
-DNA fragment sticks to itself, rather than inserting to plasmid
how is the host cell grown
fermenter is used to grow multiple copies of the host cell which have been identified with recombinant plasmid.
-this large cloned population can then produce the protein coded for by the inserted DNA fragment e.g. bacteria producing insulin from inserted insulin gene.
stages of in-vivo cloning
-desired gene is isolated using restriction endonuclease enzymes
-promoter regions and terminator regions are added
-gene is inserted into a vector-usually a plasmid
-transformation of host cell with recombinant plasmid
-identify transformed cells
-grow the host cell (clone/make copies of the gene)
in vitro cloning
uses the polymerase chain reaction (PCR) to make millions of copies of a fragment of DNA using an automated machine.
-fragments of DNA undergo a series of heating and cooling using a thermocycler to replicate them many times in a similiar way to DNA replication in nucleus
what does the reaction mixture for PCR contain
-contains the DNA fragment to be amplified
-primers- short sequence of single-stranded DNA that are complementary to the start and end of the fragment
-free nucleotides to match exposed bases
-DNA polymerase- taq polymerase
where is taq polymerase obtained from
-the DNA polymerase used is obtained from a thermophilic bacteria. It is stable at high temperatures, optimum is 72 degrees therefore will not denature in high temps in thermocycler.
what is a thermocycler
computer-controlled machine that varies temperature precisely over a period of time
process of amplifying DNA fragments using PCR
- denaturing-temperature is first increased to 95 degrees to break hydrogen bonds and to split DNA into single strands
- annealing-mixture is then cooled to 55 degrees so that primers can anneal (bind by hydrogen bonding) to end of each single strand which identifies where taq polymerase should bind
- synthesis- temperature is increased to 72 degrees for taq DNA polymerase to catalyse the addition of free nucleotides by complementary base pairing to create 2 complete double strands DNA
- whole process begins again and is repeated for many cycles.
advantages of PCR
-automated: more efficient
-rapid: billions of copies of DNA made within hours
-doesn’t require living cells: quicker and less complex techniques needed
what are VNTRs
Variable Number Tandem Repeats (VNTRs) are repeating sequences found in introns-non-coding sequences.
-probability of two individuals having same VNTRs is very low, the more closely related-the more similiar.
genetic fingerprinting
the analysis of VNTR DNA fragments to determine genetic relationships and genetic variation within a population.
-compare two DNA samples and determine whether they are from the same individual
process of genetic fingerprinting
-DNA sample is obtained
-smaller fragments of DNA containing VNTRs are cut out using restriction endonucleases, labelled and amplified using PCR
-fragments are separated using gel electrophoresis
-Banging patterns of each DNA fragment can then be compared to known sample/ ladder
process of gel electrophoresis
-DNA samples are loaded into small wells in agar gel. The gel is placed in a buffer liquid containing an electric current
-DNA is negatively charged, so DNA samples move through the gel towards the positive end of the gel.
-agar gel creates resistance for DNA-making it move slower, smaller pieces of DNA can move faster and further along
-different lengths of DNA (VNTRs) are separated.
-an alkaline buffer is then added to separate the double strands of DNA
what are DNA probes
short, single stranded pieces of DNA complementary in base sequences to the VNTRs. The probes are radioactively or fluorescently labelled.
process of DNA hybridisation
-different DNA probes are mixed with the single stranded DNA VNTRs on the agar gel for the to bind (hybridise)
-VNTRs and DNA probes are trasferred onto a nylon sheet
-the nylon sheet can then be exposed to x-rays to visulise the position of radioactive gene probes, or UV light if fluorescent probes were used.
-the position of DNA bands are compared to identify genetic relationships, the presence of a disease causing gene and to match unknown samples from a crime scene
uses of genetic fingerprinting
-forensic science e.g. identify victims of suspects
-medical diagnosis e.g. identify type of heaemoglobin produced by an individual to diagnose sickle cell anaemia
-animal and plant breeding- ensure they not closely related before being bred-> checking pedigree (anscestory)
how can DNA probes be used to locate specific alleles
-probe is designed to be complementary to allele want to find
-labelled and amplified using PCR then added to sample of single stranded DNA
-if allele is present then probe will bind (hybridise)
-after hybridisation, the DNA is washed so any unbound DNA probes are washed away
-presences of radioactive or fluorescent label therefore indicates that allele is present
applications of DNA probes
-to screen someone’s DNA for a particular heritable health condition
-to identify a gene for use in genetic engineering
-to predict how someone will respond to a drug
personalised medicine
-can be used to target different dosages or compounds as medicine depending a person’s genotype
-by determining the best doses- increases effectiveness, safety and can save money
e.g. some breast cancer treatments are targeted at specific mutations so screening can be used to ensure right medication is used
genetic counselling
-having DNA screened is process that needs to be thought out carefully, genetic counsellors guide people to make informed decision.
-people can have their family history researched to consider likelihood of carrying alleles linked to diseases before starting a family or for their general health.
-examples include screening for cystic fibrosis/ sickle cell anaemia prior to starting a family
-if there is family history of breast cancer-can screen for alleles linked to it. If allele is present: can be screened for tumors more frequently, reduce environmental risk factors or even opt for mastectomy.
purpose of a microarray
used to screen a sample with multiple DNA probes at once
-so can be used to screen for multiple diseases simultaneously
