Genome projects and gene technology Flashcards
What does it mean to ‘sequence a genome’? (1)
To know the exact sequence of bases that make up the entire DNA of an organism
What is the proteome? (1)
Full range of proteins produced by cells
Applications of determining the proteome (1)
Allows identification of potential antigens for use in vaccine production
Why can the genome not be easily translated into the proteome in more complex organisms? (2)
Due to non-coding DNA and regulatory DNA
What is meant recombinant DNA technology? (1)
Transfer of DNA fragments from one organism to another
What does the term universal mean? (1)
The same triplet codes for the same amino acid
What does degenerate mean? (1)
More than one triplet codes for the same amino acid
What does non-overlapping mean? (1)
Each base is part of only one triplet
(a base from one triplet cannot be used in an adjacent triplet)
Why is recombinant DNA technology possible? (3)
Because the genetic code is:
degenerate
universal
non-overlapping
What are the 5 steps in recombinant DNA technology? (5)
Isolation of genes
Insertion
Transformation
Identification
Growth/cloning
How can reverse transcriptase (RT) be used to produce fragments of DNA? (4)
mRNA mixed with free DNA nucleotides and RT
Free DNA nucleotides bind to single stranded mRNA template via complementary base pairing
RT joins DNA nucleotides together to form a single stranded cDNA molecule
DNA polymerase required to make cDNA double stranded
What are the advantages of using reverse transcriptase to produce fragments of DNA? (2)
mRNA is much easier to obtain
Using mRNA isolated from cytoplasm means introns have been removed, whereas genes have introns
How can restriction endonuclease (RE) be used to produce fragments of DNA? (3)
RE hydrolyse DNA at specific recognition (base) sequences
DNA sample is incubated with the specific restriction endonuclease which hydrolyse the DNA into fragments wherever the recognition sequence appears
If the target gene has recognition sequences before and after the target gene, the fragments will contain the desired gene
Why do different restriction endonucleases hydrolyse DNA at different specific recognition sequences? (1)
Because the shape of the sequence is complementary to the enzyme active site
Recognition sequences are sometimes what? (1)
Palindromic
What does palindromic mean? (1)
The base pair read is the same in opposite direction
What happens if the recognition sequence for the selected restriction endonuclease occurs within the DNA fragment you want to isolate ? (1)
This will cut the gene and it will not code for a functional protein
When can a promoter region be added? (1)
Once a DNA fragment is isolated
What does adding a promoter region, once a DNA fragment is isolated, do? (2)
Allows transcription factor to bind
Allows the gene to be expressed once inserted into the bacterial plasmid
What does a terminator region do? (1)
Stop transcription of gene of interest
How can the gene machine be used to produce fragments of DNA? (4)
Desired nucleotide sequence fed into a computer
Synthesis of oligonucleotides
Assembly of gene - oligonucleotides are overlapped then joined together and made double stranded using polymerase chain reaction (PCR)
Gene is inserted into the bacterial plasmid
Advantages of producing DNA fragments via the gene machine (2)
Artificial genes are easily transcribed and translated by prokaryotes
As they have no introns in their DNA
What are oligonucleotides? (1)
Short sequences of nucleotides
What is a vector? (1)
A DNA carrier used to transfer foreign DNA into cells
Insertion of genes into a vector method (5)
Cut open the vector DNA using the same restriction endonuclease that was used to isolate the DNA fragments
This produces complementary sticky ends between the ends of DNA fragments and cut ends of vector DNA
Target DNA fragment anneals to vector DNA by complementary base pairing between their sticky ends
DNA ligase joins the DNA fragments and vector DNA at the sugar phosphate backbone
The new recombined DNA fragment and vector DNA is recombinant DNA
What is transformation? (1)
The process by which recombinant DNA vector is transferred into a host cell
What are recombinant organisms/transformed organisms? (1)
Host cells which take up recombinant DNA
Why is it important to identify transformed cells/organisms? (1)
Not all/some vectors take up target DNA to become recombinant
not all/some host cells become transformed by taking up recombinant vectors
What is a marker gene? (1)
Allows easy identification of cells that have taken up a genetically transformed plasmid
How to identify transformed bacteria using antibiotic resistance gene (3)
Some cells will not taken up any plasmid - they are killed by both types of antibiotic
Some cells take up ‘original’ plasmid - resistant to both types of antibiotic
some cells take up ‘transformed’ plasmid - resistant to one type of antibiotic but not the second
Outline a method for in vivo gene cloning (6)
Cut the desired gene using restriction endonuclease
Make artificial DNA with correct sequences of bases using DNA polymerase
Cut plasmid open using same restriction endonuclease
Produce sticky ends
Use DNA ligase to join
Return plasmid to bacterial cells
What is the polymerase chain reaction (PCR) used for? (1)
To amplify a single fragment of DNA
What are the 3 stages of PCR? (3)
Separation
Annealing
Synthesis/elongation
What is a primer? (3)
Short pieces of single stranded DNA with complementary base sequences to the start of the DNA fragment
Prevent strands sticking back together (annealing)
Allow DNA polymerase to attach and join nucleotides together
Describe the process of PCR (5)
Heat DNA to 95 which breaks hydrogen bonds
Add primers and add DNA nucleotides
Cool to 50 to allow binding of nucleotides
Add DNA Taq polymerase
Heat to 75
DNA polymerase joins nucleotides together
Repeat cycle many times
Advantage of using in ‘vivo’ compared to in ‘vitro’ (2)
In vivo can be used to produce mRNA or proteins from inserted DNA as well as target DNA
In vitro can only be used to copy DNA
Advantage of using in ‘vivo’ compared to in ‘vitro’ in terms of errors (2)
In vivo more accurate because cells have mechanisms for correcting errors that are made when copying genes
In vitro lacks error correcting mechanisms so the error rate is higher than cell based methods
Why is there almost no risk of contamination in vivo (2)
Pure samples
and can be used to produce large quantities of gene products
Medical applications in recombinant DNA technology (2)
Produce a faulty or lack of protein (insulin)
Gene therapy to correct for a mutation or lack of gene expression
Agriculture applications in recombinant DNA technology (4)
Produce crops with larger yields or that are resistant to disease
Help meet rising food demands
Help plants survive changing environmental conditions
Reduce environmental problems associated with spraying pesticides
Benefit of recombinant DNA technology in biological processes (1)
Regulation of gene expression, DNA replication, mitosis, and cell death
Benefit of recombinant DNA technology in designing new or improving existing industrial processes (1)
Produce large quantities quickly and cheaply
Concerns regarding recombinant DNA technology (2)
Antibiotic resistance may spread owing to its use as a selection marker
Inserting new genes into a crop plant could disrupt other genes/functions creating toxic products within genetically modified food sources
What are liposomes?
Lipid droplets which can cross the phospholipid bilayer and release target DNA into the cell
Difference between somatic gene therapy and germ line therapy (2)
Somatic is when DNA is transferred to our normal body tissue
Germ line is when DNA is transferred to cells that produce eggs or sperm
What is DNA hybridisation and what is the purpose of it? (2)
When the DNA probe binds to its complementary target DNA
Purpose: measure the degree of difference between two strands of DNA. Can be used to compare someone’s DNA to a certain gene to see if they have it
What is a gene probe? (1)
A short, single stranded DNA molecule with a complementary base sequence to DNA fragment to be located which is radioactive or labelled by a florescent molecule
What are variable number tandem repeats? (1)
Region of DNA between genes
What is gel electrophoresis? (1)
Analysis of difference in the length of VNTRs
Describe how genetic fingerprinting is carried out (9)
DNA extracted from sample
DNA cut using restriction endonucleases
Must leave VNTRs intact
DNA fragments separated using gel electrophoresis
Mixture put into wells on gel and electric current passed through
Immerse gel in alkaline solution
Cover with nylon
DNA fixed to nylon using UV light
Radioactive marker/probe added
Areas with probe identified using X-ray film
Explain why plant cells that have the plasmid DNA inserted into plant DNA nucleus grow into plants where all cells contain the desired gene but plant cells that have the plasmid DNA inserted into their cytoplasm grow into plants that do not contain the desired gene (3)
(DNA of desired gene) replicated with host DNA/inside nucleus
Passed on by mitosis/plant grows by mitosis
Produces genetically identical cells
Limitations of somatic gene therapy (5)
Not all cells take up new DNA
Not all cells express DNA allele
Only some tissue types are accessible i.e., lungs, needs to be repeated – as cells die replaced by cells with faulty DNA,
Multiple treatments may be needed
Body can produce an immune response to the vector
Application of genome sequencing (3)
Comparing genomes between species to determine evolutionary relationships
Genetic matching
Personalised medicine
Synthetic biology
How can DNA probes be used to locate specific alleles? (3)
The probe is designed so that its sequence is complementary to the allele
They are labelled, amplified using PCR, then added to a sample of single stranded DNA
The probe will bind if the allele is present
Applications of DNA probes (3)
Use to screen DNA for particular heritable health conditions
To identify a gene for use in genetic engineering
To predict how someone will respond to drugs
