3.8.4 gene technologies

Question 1

what techniques have been developed to produce pure proteins

Answer 1

to isolate genes, clone them and transfer them to into microorganisms, which are then grown to provide a factory for the continuous production of the desired protein

Question 2

what is recombinant DNA

Answer 2

the DNA of 2 different organisms that has been combined in the way of isolation, cloning and transferring
the resulting organism is known as a transgenic or genetically modified organism (GMO)

Question 3

how does the DNA function normally in other organisms

Answer 3

the genetic code is the same in all organisms. it is universal and can be used by all living organisms.
the making of proteins is also universal as the mechanisms of translation and transcription are essentially the same in all living organisms

Question 4

what is the process of making a protein using DNA technology of gene transfer and cloning

Answer 4

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

Question 5

how to produce DNA fragments

Answer 5

conversion of mRNA to cDNA
using reverse transcriptase
using restriction endonucleoases to cut fragments containing the desired gene from DNA
creating the gene in a gene machine, usually based on a known protein structure

Question 6

how do you use reverse transcriptase to convert

Answer 6

coded genetic information pf retroviruses is in the form of RNA
In a host cell, they are able to synthesise DNA from RNA using an enzyme called reverse transciptase
reverse transcriptase catalyses the production it DNA to RNA

Question 7

what is the use of reverse transcriptase to isolate the gene that codes for insulin ( the process)

Answer 7

1. B cells from islet of langerhans. as they are specialised for insulin so make lots of mRNA that codes for protein
2. mRNA acts a template on which a single-stranded complementaary copy of DNA (cDNA) is formed ysing reverse transcriptase
3. cDNA is isolated by hydrolysis of mRNA with an enzyme
4. double-stranded DNA is formed on template of cDNA using DNa polymerase

Question 8

how do you use restriction endonucleases

Answer 8

they cut a DNA double strand at a specific sequence of bases called a recognition sequence. sometimes, this occurs between 2 opposite base pairs to leave 2 straight edges known as the blunt ends.

Question 9

how does restriction endonucleases make blunt ends

Answer 9

sometimes, this occurs between 2 opposite base pairs to leave 2 straight edges known as the blunt ends

Question 10

how does restriction endonucleases make sticky ends

Answer 10

cut DNA in a staggered fashion to leave an uneven cut where each strand of DNA has exposed unpaired bases

Question 11

what is a palindrome

Answer 11

if you read 4 unpaired bases at each end from left to right, the 2 sequences are opposites of each other

Question 12

what are restriction endonucleases

Answer 12

bacteria defend themselves by producing enzymes that cut up viral DNA. these enzymes are restriction endonucleases

Question 13

what is the gene machine

Answer 13

manufacturing of genes

Question 14

what is the process of manufacturing genes from the gene machine

Question 15

what is invivo gene cloning

Question 16

how you prepare the DNA fragment for insertion

Answer 16

addition of extra lengths of DNA.
for the transcription of any gene to take place, the enzyme that synthesises mRNA much attach to DNA near a gene
binding site for RNA polymerase is a promoter region

Question 17

how do you insert DNA fragment into a vector

Answer 17

once an appropriate DNA fragment has been cut and the promoter and terminator regions added, the next step is to add to a carrying region (vector)

Question 18

how do vectors work

Answer 18

transports DNA into a host cell

there are different types of vectors but most commonly used is the plasmid

Question 19

what are plasmids

Answer 19

circular lengths of DNA, found in bacteria, which are separate from their main bacterial DNA
almost always contain genes for antibiotic resistance and restriction endonucleases are used at 1 of these antibiotic-resistance genes to break the plasmid loop

Question 20

how do you introduce DNA to host cells

Answer 20

once the DNA has been incorparated into at least some of the plasmid, they must be reintroduced into bacterial cells.
this process is called transformational and involves the plasmid and bacterial cells being mixed together in a medium containing calcium ions

Answer 21

the calcium ions and changes in temperatures make the bacterial membrane permeable, allowing the plasmid to pass through the cell-surface membrane, allowing the plasmids to pass through the cell-surface into the cytoplasm.
not all of the bacterial cells will possess the DNA fragment with the desired genes for the desired protein. there are reasons

Question 22

what are the reasons for not all of the bacterial cells not possessing the DNA fragments with the desired gene

Answer 22

• only a few bacterial cells ( as few as 1%) take up the plasmids when the 2 are mixed together
• some plasmids will have closed up again without incorporating the DNA fragments
• sometimes the DNA fragment ends join together to form its own plasmid

Question 23

what is the first task in regards to introduction

Answer 23

identify what bacterial cells take up the new plasmid.

Question 24

how to identify what bacterial cells have taken up the plasmid

Answer 24

use the gene for anitbiotic resistance , which is unaffected by the introduction of the new gene

Question 25

the process of finding what bacterial cells has taking up the plasmids

Answer 25

all bacterial cells are grown on a medium that contains the antibiotic
bacterial cells that have taken the plasmid will have acquired the gene for resistance
the bacterial cells are able to break down the antibiotic and therefore survive
the bacterial cells that haven’t taken the plasmids will die

Question 26

What are marker genes

Answer 26

there are a number of ways of using marker genes to identify whether a gene has been taken up
all involve using a second, separate gene on the plasmid. the 2nd gene is easily identifiable for 1 reason

Question 27

what are reasons the 2nd gene is unidentifiable

Answer 27

• may be resistant to an antibiotic
• may make a fluorescent protein that is easily seen
• may produce an enzyme whose action can be identified

Question 28

what are antibiotic-resistance marker genes

Answer 28

old technology and has been superseded by other methods

[see physical flashcards for the process]

Question 29

what are fluroscent markers

Answer 29

transfer of genes from jellyfish into the plasmid
the gene in question produces a green fluoroscent protein.
the gene to be cloned is transplanted into the centre of GFP
any bacterial cell that has been taken up the plasmid with the gene that is to be cloned cant produce GFP and not flurocse

Question 30

what are enzyme markers

Answer 30

a 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 a plasmid with the required gene is present in a bacterial cell, the colonies formed from it wont produce lactase
so when the bacteria cell are grown on the colourless substrate, there are unable to change colour

Question 31

what are polymerase chain reaction

Answer 31

PCR method
a method of copying fragments of DNA
is automated, making it rapid and efficient

Question 32

what does the process of PCR require

Answer 32

DNA fragment is to copied
DNA polymerase- obtain taq polymerase is obtained from hot springs (tolerant to heat, no denature)
primers- short sequence of nucleotides that has a set of bases complementary to those at either end
nucleotides
thermocyclers- computer-controlled machine that varies temperature precisely over a period of time

Question 33

what is the stages of the polymerase chain reaction

Answer 33

1. separationn of the DNA strand- DNA fragments, primers, and DNA polymerase are placed in a vessel of a thermocycler. temperature increases to 95, causing 2 strands to separate
2. addition of the primers. cooled to 55, causing the primer to join to complementary bases.
3. synthesis of DNA. - temp increase to 72. optimum temp for DNA polymerase to add complementary nucleotides along each of the separated DNA strands

Question 34

what are the advantages of in vitro gene cloning

Answer 34

extremely rapid- within a matter of hours, 100 billion of a gene can be made. will massively increase any contaminating DNA found.
doesn’t require living cells.- all it needs is a base of DNA that needs amplifying

Question 35

what is the advantages of in vivo gene cloning

Answer 35

particularly useful when we wish to introduce a gene into another organism.
no risk of contamination
very accurate
cuts specific genes
produces transformed bacteria that can be used to produce large amounts of gene products

Question 36

what are DNA probes

Answer 36

a short single stranded length of DNA that has some sort of label attached that makes it easily identifiable.
2 most common probes:
1. radioactive labelled probes- made up of nucelotide of isotope 32p. identified by X-ray film exposed by radioactivity
2. flurescently labelled probes- emit light

Question 37

DNA probes are used to identify particular alleles of genes

Answer 37

• DNA probe is made that has base sequence that are complementary to part of the base sequence that makes up the allele of the gene
• double-stranded DNA that is being tested to separate its 2 strands
• separated DNA strands are mixed with the probes, which binds to the complementary base sequence (DNA hybridisation)

Question 38

what is DNA hybridisation

Answer 38

takes place when a section of DNA/RNA is combined with a single-stranded section of DNA which has complementary bases
2 strands must be separated first. achieved by heating DNA until its double strand separates into 2 strands. when cooled, they recombine (anneal) with each other to reform the other.

Question 39

how di you locate specific alleles of genes

Answer 39

determine the sequence of nucleotide bases of the mutant allel. achieved via sequencing techniques.
fragment of DNA is produced that has a base sequence complementary to mutant allele
multiple copies of probe are formed via polymerase chain reaction
probe is made by attaching a marker
hybridisation

Question 40

what is genetic screening

Answer 40

many genetic disorders are the result of gene mutations

Question 41

what is the summary of the process to locate a specific allele

Answer 41

[see physical flashcard for process]

Question 42

what is personised medicine

Question 43

what is genetic counselling

Question 44

what is genetic fingerprinting

Question 45

what is gel electrophoroesis

Question 46

what is the summary or genetic fingerprinting technique

Question 47

how to use genetic fingerprinting use in genetic relationships and variability

Question 48

how tp use DNA fingerprinting in forensic science

Question 49

how to use DNA fingerprinting in medical diagnosis

Question 50

how to use DNA fingerprinting in plant and animal breeding