Recombinant DNA technology

Question 1

What is the process of making a protein using DNA technologies

Answer 1

1. isolation
2. insertion
3. transformation
   4.identification
   5.growth and cloning

Question 2

What are the methods of producing a DNA fragment

Answer 2

-using reverse transcriptase
-restriction endonuclease
-gene machine

Question 3

How are DNA fragments produced using restriction endonuclease

Answer 3

-DNA is cleaved at a restriction site
-restriction endonuclease cuts out the DNA fragments
-staggered cuts leave sticky ends which are several nucleotide bases long
-Any piece of DNA cut with the same restriction site will have complimentary sticky ends
-Complimentary bases of the sticky ends pair up
-DNA ligase joins sugar phosphate backbone
-Allows us to join DNA from one organism to another

Question 4

How are DNA fragments produced using reverse transcriptase

Answer 4

-Strand of mRNA from the beta cells of human pancreas coding for the production of insulin
-mRNA acts as a template for the production of a single stranded complimentary copy of cDNA using reverse transcriptase
-cDNA is isolated by hydrolysis of the mRNA with DNA helicase
-Double stranded DNA is formed on the template of the cDNA using DNA polymerase resulting in a copy of the human insulin gene

Question 5

How are DNA fragments produced using the gene machine

Answer 5

-Desired protein with amino acid sequence, mRNA sequence and DNA sequence
-nucleotide base sequence into computer
-biosafety, biosecurity and ethics checked
-computer designs oligonucleotides
-oligonucleotides assembled one nucleotide at a time
-oligonucleotides joined to form gene
-PCR replicates gene
-gene into plasmid using sticky ends
-gene check using sequencing techniques

Question 6

What does in vivo mean

Answer 6

transferring the fragments to a host cell using a vector

Question 7

What does in vitro mean

Answer 7

using the polymerase chain reaction

Question 8

Describe the process of in vivo gene cloning

Answer 8

-DNA fragment is produced from restriction endonuclease
-RNA polymerase and transcription factors binds to the promoter region of the DNA fragment
-Terminator is added to stop transcription at appropriate time
-Same restriction endonuclease used to cut fragment is used to cut plasmid at antibiotic resistant gene which produces complimentary sticky end
-plasmid is transformed into the bacteria using calcium irons and changing the temperature
-identify uptake of gene/plasmid using R plasmid which carry the gene for resistance to two antibiotics, ampicillin and tetracycline
-Determine if the gene is present using a marker gene

Question 9

Describe the process of PCR

Answer 9

-DNA fragments, primers and DNA polymerase placed in thermocycler and heated to 95 to break hydrogen bonds separating the strands
-cool to 55 so primers can anneal to DNA at complimentary bases
-heat to 72 so free nucleotides can bind to complimentary base pairs and DNA polymerase can form phosphodiester bonds

Question 10

What are the advantages of in vitro cloning

Answer 10

-it’s extremely rapid
-it does not require living cells

Question 11

What are the advantages of in vivo cloning

Answer 11

-it is particularly useful where we wish to introduce a gene into another organism
-it involves almost no risk of contamination
-it’s very accurate
-it cuts out specific genes
-it produces transformed bacteria that can be used to produce large quantities of gene products

Question 12

What does PCR require

Answer 12

-DNA fragment
-DNA polymerase
-primers
-nucleotides
-thermocycler

Question 13

What is a primer

Answer 13

Short sequence of nucleotides that have a set of bases complimentary to those at one end of each of the two DNA fragments

Question 14

What is a thermocycler

Answer 14

A computer controlled machine that varies temperatures precisely over a period of time

Question 15

DNA probe

Answer 15

short single stranded length of DNA that has a label attached to it that makes it easily identifiable

Question 16

How are DNA probes used

Answer 16

-Isolate DNA from bodily fluid sample
-denature the single strand of DNA
-combine with DNA probes that are complimentary
-DNA probes will bind to the gene of interest if it’s present in the sample

Question 17

What are the two most common types of DNA probe

Answer 17

-Radioactively labelled probes
- fluorescently labelled probes

Question 18

How are radioactive DNA probes identified

Answer 18

Using X-ray film that is exposed by radioactivity

Question 19

How are fluorescent DNA probes identified

Answer 19

Emit light under certain conditions, for example when the probe has bound to the target DNA sequence

Question 20

DNA hybridisation

Answer 20

To 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

Question 21

How are specific alleles located using DNA probes and DNA hybridisation (mutant allele that causes disease)

Answer 21

-Determine the sequence of nucleotide bases of the mutant allele
- Make a DNA probe that has bases complimentary to the mutated gene
-Separate the double stranded DNA from the sample to make it single stranded using DNA helicase or heat
-Mix separate strands of DNA sample with DNA probe and cool
-DNA probe will bind to the complimentary base sequence of the mutated gene on the DNA sample (DNA hybridisation)
-wash away unattached DNA probes
-site where the probe has bound is identified by either the radioactivity or fluorescence that the probe emits

Question 22

genetic counselling

Answer 22

allows people to make informed decisions about themselves or their offspring by providing advice and information

Question 23

genetic screening

Answer 23

can determine the probability of a couple having offspring with a genetic disorder

Question 24

genetic fingerprinting

Answer 24

process that relies on the fact that the DNA of individuals is unique and contains repetitive non-coding bases called VNTRs

Question 25

What are VNTRs

Answer 25

They are non-coding and inherited from parents. They are unique in every indivual expect twins

Question 26

Gel electrophoresis

Answer 26

used to separate DNA fragments according to size

Question 27

How does gel electrophoresis work

Answer 27

-DNA fragment is placed on agr gel and voltage is applied along it
-resistance of the gel means that the larger the fragments, the more slowly they move
-over a period of time the smaller fragments mover further
-If the fragment is labelled with a probe it can be determined with a x-ray sheet

Question 28

What are the 5 stages of making a genetic fingerprint

Answer 28

-extraction
-digestion
-separation
-hybridisation
-development

Question 29

How are the 5 stages of genetic fingerprinting carried out

Answer 29

-DNA is extracted from the sample
-Restriction endonucleases cut the DNA into fragments
-They are separated using gel electrophoresis and transferred to from the gel to nylon membrane
-DNA probes are added to label the fragments and they attach to specific fragments
-membrane with radioactively labelled DNA fragments is placed onto an X-ray film

Question 30

What are the uses of DNA fingerprinting

Answer 30

-crime scene investigation
-resolve questions of paternity
-medical diagnosis

Question 31

How is DNA accepted and functioning by different species

Answer 31

the genetic code is universal

Question 32

palindromic

Answer 32

when you read the unpaired bases both from left to right the two sequences are opposites of one another

Question 33

What are advantages of using the gene machine

Answer 33

-any sequence of nucleotides can be produces in a short time
-accurate
-the artificial genes are free of introns and other ‘non’ coding DNA

Question 34

What is the importance of sticky ends

Answer 34

Complimentary sticky ends from different organisms can be joined using DNA ligase

Question 35

How do R-plasmids work in in vivo gene cloning

Answer 35

-all the bacteria cells are grown on a medium containing ampicillin
-bacterial cells that have taken up the plasmid will have acquired the gene for resistance
-these bacterial cells are able to break down the ampicillin and survive
-the cells that have not taken up he plasmids will not be resistant and die

Question 36

Marker genes

Answer 36

Involve using a second, separate gene on the plasmid which is easily identifiable

Question 37

Why are marker genes easily identifiable

Answer 37

-May be resistant to an antibiotic
-May produce a fluorescent protein that is easily seen
-Produce an enzyme whose actions can be identified

Question 38

Antibiotic resistance marker genes

Answer 38

replica plating
-requires tetracycline which was cut
-no longer produce the enzyme that breaks it down
-no longer be resistant
-can therefore identify these bacteria by growing them on a culture that contains tetracycline

Question 39

Fluorescent markers

Answer 39

-transfer of a gene from a jellyfish
-produces a green fluorescent protein (GFP)
-Any bacterial cell that has taken up the plasmid with gene that is to be cloned will not be able to produce GFP
-Results can b obtained by looking under a microscope

Question 40

vector

Answer 40

transfers genes from one organism into another

Question 41

Enzyme markers

Answer 41

-gene that produces the enzyme lactase
-lactase will turn a particular colourless substrate blue
-required gene is transplanted into the gene that makes lactase
-If the plasmid with the required gene is present it won’t produce lactase- won’t change colour

Question 42

polymerase chain reaction

Answer 42

method of copying fragments of DNA