recombinant DNA technology Flashcards
Explain how complementary DNA is made using reverse transcriptase
-Strand of mRNA from the cells that produce the desired protein
-mRNA acts as a template for production of a single stranded complementary copy of DNA (cDNA) using reverse transcriptase
-cDNA is isolated by hydrolysis of the mRNA with an enzyme
-Double stranded DNA is formed on the template of the cDNA using DNA polymerase
-This is the gene for the protein
Explain how restriction endonucleases are used to cut DNA into fragments
-Restriction endonucleases cut DNA at specific base sequence (recognition site)
-breaking phosphodiester bonds
-This produces ‘sticky ends’ (short unpaired sequence of bases)
-Sticky ends will be complementary to the sicky ends on other DNA cut with the same restriction endonuclease
Define recombinant DNA technology
process by which genes are altered, manipulated and transferred between organisms
What is recombinant DNA
DNA of two different organisms that has been combined
Explain why it is possible to transfer DNA between organisms and for proteins to still be produced
the genetic code and protein production process are universal
Explain how the gene machine is used to produce DNA fragments
-Desired protein
-Amino acid sequence
-mRNA sequence/triplets
-DNA sequence
-Nucleotide base sequence into computer for Biosafety/biosecurity/international standards/ethics check
-Computer designs oligonucleotides (small, overlapping single strand nucleotides)
-Oligonucleotides assembled one nucleotide at a time (automated)
-Oligonucleotides joined to form gene (all coding DNA. Single strand)
-PCR replicates gene (makes complemetary strand for DNA first)
-Gene into plasmid using sticky ends (vector for storage/cloning/transfer)
Genes check using sequencing techniques
State the stages in making a protein using DNA technology
isolation, insertion, transformation, identification, growth/cloning
State 3 ways to isolate a fragment of DNA
reverse transcriptase, restriction endonucleases, gene machine
What is the function of reverse transcriptase?
to produce DNA from RNA
Explain how DNA is made using reverse transcriptase
isolate mRNA –> reverse transcriptase makes single strand of cDNA complementary to mRNA –> isolate single strand of cDNA by hydrolysis –> DNA polymerase forms double stranded DNA
What is a restriction endonuclease?
enzyme that cuts a strand of DNA at a specific base sequence (recognition site)
What is a sticky end?
short sequence of unpaired bases left after DNA is cut with restriction endonuclease
Describe the purpose of the gene machine
to produce a double stranded gene from a know base sequence
Describe how a DNA base sequence can be determined from a protein
protein –> amino acid sequence –> mRNA codons –> complementary DNA base sequence
What is a a DNA base sequence checked for before production in a gene machine?
biosafety and biosecurity
Name the short sections of DNA produced in the gene machine
oligonucleotides
How is the order of the oligonucleotides in the gene determined?
the sections overlap
State two ways to clone genes
-in vivo
-in vitro
Explain why it is important to use the same restriction endonuclease to cut DNA from different organisms that you wish to combine
the restriction endonuclease will make a specific sticky end and this must be complementary to that on the other piece of DNA
Name the enzyme used to to join the sugar phosphate backbone of recombinant DNA
DNA ligase
Describe how to prepare a DNA fragment for insertion
attach a promotor (for RNA polymerase to bind to) and a terminator (to stop transcription)
Explain the importance of sticky ends
Short unpaired sequence of bases produced when DNA is cut by a restriction endonuclease. Allow us to bind DNA from one organism to another provided the same restriction endonuclease is used
Explain how a DNA fragment can be inserted into a vector
-Target DNA and plasmid cut with the same restriction endonuclease so have complementary sticky ends which base pair.
-Phosphodiester bonds formed using DNA helicase
Explain how the DNA of the vector is introduced into host cells
-Transformation
-Calcium ions and change in temperature are required to make cell walls more permeable
Describe the nature of gene markers and explain how they work
-Marker genes are genes in a plasmid that code for antibiotic resistance/fluorescent protein
They can be used to detect whether:
a) a bacterium has taken up a plasmid (bacteria containing the plasmid will be resistant to antibiotic/will glow)
b) the plasmid has taken up the desired gene (desired gene inserted into antibiotic resistance/fluorescence/enzyme gene - bacteria that are not resistant to antibiotic/do not glow/do not catalyse reaction contain the desired gene)
Describe the polymerase chain reaction
In vitro gene cloning. Automated process of copying fragments of DNA
Explain how the polymerase chain reaction is carried out
-Requires DNA fragment, DNA polymerase, (DNA) nucleotides and primers;
-Heat to 95 °C to break hydrogen bonds (and separate strands);
-Reduce temperature to 55 °C so primers bind (anneal) to DNA at their complementary bases.
-This provides a starting point for DNA polymerase to bind;
-Increase temperature to 72 °C, optimum temperature for DNA polymerase to join nucleotides;”
Summarise the advantages of in vivo and in vitro cloning
In vitro:
-Rapid
-Does not require living cells
In vivo:
-Useful to introduce genes into other organisms
-No contamination
-Accurate
-Cuts out specific genes”
Describe what DNA probes are and explain how they work
DNA probe is a short single strand of DNA labelled with either a radioactive or fluorescent marker, with bases complementary with required gene;
-Used to identify specific alleles of a gene by binding a detecting”
Explain how DNA hybridisation is used to locate specific alleles of genes
-DNA hybridisation: when a section of DNA or RNA binds with a single stranded section of DNA with complementary bases.
-To locate gene:
-Make DNA sample single stranded
-The probe will bind to specific gene;
-Use autoradiography/x ray film/detect fluorescence to show the bound probe;”
Describe the use of labelled DNA probes to screen for heritable conditions or health risks
DNA probes can be used to identify alleles of genetic disorders or that predispose people to diseases
Consider the use of genetic screening in genetic counselling
Genetic screening allows people to make informed decisions during genetic counselling about themselves or their offspring
Describe what genetic fingerprinting is
Process that relies on the fact that the DNA of individuals is unique and contains repetitive, non coding bases called VNTR’s (variable number tandem repeats)
Explain the technique of gel electrophoresis
-Used to separate fragments of DNA in size order
-DNA fragments on agar gel with voltage across it.
-Negatively charged DNA moves towards the anode (positive)
-Larger the fragment the slower the movement
-Therefore less distance travelled in a fixed time
-Fragments labelled with DNA probes
-Determine final position on gel by applying x ray film on the gel
-Radioactivity from the DNA exposes the film and maps the fragments”
Explain how genetic fingerprinting is carried out
-Extraction- extract DNA from sample
-Digestion - Restriction endonucleases cut DNA into fragments
-Separation - Separate fragments using gel electrophoresis and transfer from gel to nylon membrane
-Hybridisation - Add DNA probes to label DNA fragments
-Development - Nylon membrane with DNA fragments placed on X ray film, development of the film reveals dark bands where the probes have attached”
Explain how the results of genetic fingerprinting are interpreted
-Visually compare samples and if similar use automated scanning machine which which calculates and compares DNA fragment length based on distance travelled of known samples. -Closer the match in sample patterns the more likely the samples were from the same person
Consider the uses of genetic fingerprinting
-Genetic relationships - paternity case resolution, bands on children’s genetic fingerprint will each match with one of their parents
-Genetic variability - within populations, similar genetic fingerprints means little genetic diversity
-Forensic science - DNA at the scene of a crime can be analysed to determine someone’s presence at the crime scene
-Medical diagnosis - compare genetic fingerprints of patients with those of known cases
-Plant and animal breeding - prevent inbreeding in breeding programmes or identify desirable genes
