6.3 manipulating genomes

Question 1

define genome

Answer 1

the complete genetic make up of an organism

Question 2

define DNA sequencing

Answer 2

a technique allowing genes to be isolated and read

Question 3

what did early DNA research show

Answer 3

structure of DNA was known
base triplet sequences coded for amino acids
but you couldn’t work out the sequence of nucleotide base triplets

Question 4

explain Fred Sanger’s DNA sequencing approach

Answer 4

use a single strand of DNA as a template for 4 experiments in seperate dishes containing
one of the 4 different terminators used (modified nucleotides of ATCG)
normal nucleotides
a primer
DNA polymerase
DNA fragments of various lengths were made and pass through a gel by electrophoresis
sorted by length (shorter travelled further)
bases get read off what terminator it had and it creates the sequence

Question 5

explain how DNA gets cloned

Answer 5

gene isolated using enzymes from bacteria
inserted into bacterial plasmid (vector) and then into an E coli bacterium host
when cultured divided lots
plasmid in DNA copied many times
each new bacteria contains DNA

Question 6

what technique is used in high throughput sequencing to sequence genomes

Answer 6

pyrosequencing

Question 7

what is pyrosequencing

Answer 7

synthesisng a single strand of DNA complementory to the strand to be sequenced, by one base at a time whilst detecting the light admission

Question 8

explain the process of pyrosequencing

Answer 8

DNA cut into fragments using a nebuliser
lengths are degraded into single strands of DNA
sequencing primer is added
DNA is incubated with DNA polymerase, ATP sulfurylase, luciferase, apyrase, luciferin and APS
activated nucleotides (TTP, ATP, CTP, GTP) are added one at a time
light detected

Question 9

what is the human genome project

Answer 9

project launched to sequence the human genome
contains about 24000 genes

Question 10

how does the human genome project work

Answer 10

DNA samples are collected and these samples are sequenced and compared to create a reference genome
multiple copies of DNA collected from people

Question 11

how has the human genome project been used

Answer 11

tackle human health issues by finding cures
correlation between changes in specific genes and the likelihood of developing certain inherited diseases

Question 12

how can genome wide comparisons be made between species

Answer 12

whole genome sequencing determins the complete DNA sequence of an organisms genome

Question 13

how can genome wide comparisons be made between individuals

Answer 13

all have same genes but different alleles
mutations can cause difference between DNA sequences

Question 14

how can DNA sequencing be used to look at genetic variation and evolutionary relationships

Answer 14

genomes of individuals sequenced and compared
species with a high level of genetic variation will exhibit a large number of difference in base sequences between individuals

species with fewer differences in genomes are likely to share a common ancestor
eg. protein cytochrome c

Question 15

how can DNA sequencing be used to look at genotype and phenotype relationships

Answer 15

relationships are explored by knocking out different genes by stopping their expression and observing the effect it has on the phenotype of an organism

Question 17

what is bioinformatics

Answer 17

developing and using computer software that can analyse / store biological data

Question 18

how can we predict the amino acid sequence of proteins

Answer 18

can work out the base triplets which code for an amino acid = can determine the orimary structure of an amino acid

Question 19

define intron

Answer 19

non coding bit of DNA that doesn’t code for an amino acid

Question 20

define gene

Answer 20

sectionof DNA that codes for a protein and results in a characteristic

Question 21

define locus

Answer 21

position of a gene on a chromosome

Question 22

what is synthetic biology

Answer 22

involves designign and building biological dveices and systems

Question 23

uses of synthetic biology

Answer 23

information storage onto single strands of DNA
productionof medicines
development of porotiens - eg. haemoglobin
biosensors
nanotechnology

Question 24

what is DNA profiling

Answer 24

technique used to determin patterns in the non coding DNA of an individual

Question 25

explain the process of DNA profiling

Answer 25

DNA is obtained from the individual digested with enzymes into fragments fragments are sperated by gel electrophoresis a banding pattern is created and can be compared

Question 26

who developed DNA profiling

Answer 26

Alac Jeffreys

Question 27

uses of DNA profiling

Answer 27

forensic science maternity and paternity disputes analysis of disease

Question 28

define polymerase chain reaction

Answer 28

technology that can amplify a short length of DNA to lots of copies

Question 29

process of PCR

Answer 29

DNA mixed with DNA nucleotides, primers, magnesium ions and enzyme Taq DNA polymerase heated to 95 degrees c - breaks hydrogen bonds between complementary nucleotide base pairs into two single strands cooled to 68 degrees c - primers can bind to one end of each single strand (gives a small section of a double strand) DNA polymerase enzymes bind to the end where the primer is heated to 72 degrees c - optimum temp for DNA poly DNA poly catalyses nucleotides joining the single strand in the 5' to 3' direction new double strand generated repeats cycles

Question 30

uses of PCR

Answer 30

forensics - small quantities of DNA can be amplified analysis of disease risk - detecting mutations identify viral infections tissue typing - reduces risk of rejection of transplants detection of cancer research of extinct species

Question 31

define electrophoresis

Answer 31

process used to seperate proteins/ DNA fragments of different sizes

Question 32

explain how electrophoresis works

Answer 32

extraction of DNA from sample fragments are created using restriction endonucleas enzymes) seperation of fragments by running electric current over it (attracted to positive electrode at different rates) in an agarose gel and buffer solution smaller fragments = travel further fluorescent dye/ probes used to show

Question 33

how can electrophoresis be used for seperating proteins

Answer 33

charged solution to make all proteins have same charge proteins seperate as they move through the gel according to molecular mass

Question 34

what is a DNA probe

Answer 34

short single stranded length of DNA complementory to section of DNA being investigated has fluorescent dye/ radioactive tag

Question 35

explain how a DNA probe is used

Answer 35

DNA probe binds to the complemenotry section of the target DNA the fuorescent dye tag is shown up when UV light passes over/ radioactive tag shows up dark when exposed to photographic film identifies presence/ location of the traget DNA sequence

Question 36

uses of DNA probes

Answer 36

locate specific gene identify same gene in variety of genomes idenitfy presence of an allele

Question 37

what is genetic engineering

Answer 37

recombination DNA technology combining DNA from different organisms by isolating them and inserting them into another organism using vectors

Question 38

name the stages of DNA engineering

Answer 38

obtaining required gene placing gene into a vector get vector into recipient cell recipient expresses gene

Question 39

how is the required gene obtained in genetic engineering

Answer 39

mRNA from cells where gene is expressed is obtained enzyme reverse transcriptase catalyses formation of single strand of complementary DNA primers and DNA poly. added = makes into a double strand which codes for specific protein DNA probe used to locate a gene = cut out using restriction enzymes

Question 40

alternative ways to obtain gene in genetic engineering

Answer 40

nucleotide sequence known - gene synthesised using an automated polynucleotide synthesiser or PCR primers to amplify the gene

Question 41

explain how genes are placed into a vector in genetic engineering

Answer 41

plasmids from bacteria are mixed with restriction enzymes = cuts plasmid at specific recognition sites cut ends are sticky ends (unpaired) nucleotide bases are added = complementary to sticky ends of plasmid DNA ligase catalyses annealing (binding) of gene and cut plasmid

Question 42

alternative way to place gene into vector

Answer 42

gene placed into attenuated virus and carry it into host cell

Question 43

explain the ways that vectors are placed into recipient cell

Answer 43

heat shock treatment - making walls/ membranes of bacteria more porous electroporation - high voltage pulse applied to cell to disrupt the membrane electrofusion - electrical fields used to introduce DNA into cells Transfection - DNA packaged into bacteriophage , transfects the host cell gene placed in attenuated v. carried into host cell gene gun - small pieces of gold used, coated in DNA, fired into plant cells

Question 44

define restriction endonuclease enzymes

Answer 44

cut DNA at certain recognition points

Question 45

define vector

Answer 45

carry/ insert DNA into host organism

Question 46

define sticky ends

Answer 46

single stranded ends of DNA that has been created by endonuclease enzymes

Question 47

DNA ligase

Answer 47

joins DNA fragments joins sugar and phosphate groups of DNA backbone

Question 48

ethical issues with genetic manipulation

Answer 48

microorgansims -escape, antibiotic resistane - pathogens/virus may be inserted into genome which increases risk of cancer plants - super weeds created eg. resistance of herbicides in soya and weeds getting destroyed - concerns about eating genetically modified food - no choice whether to but gm seeds pharming - using genetically modified animals to make human proteins

Question 49

Somatic cell gene therapy

Answer 49

Gene therapy by inserting alleles into body cells Doesn't pass to offspring

Question 50

Germ line therapy

Answer 50

Gene therapy by inserting alleles into gametes/zygotes Offspring may inherit gene

Question 51

basic principle of gene therapy

Answer 51

Inserting a functional allele of a particular gene into cells containing mutated/non functioning alleles of the gene

Question 52

How can a liposome be is used to insert genes

Answer 52

Alleles are packaged into the liposome 'sphere of lipid bilayer' Sprayed by an aerosol into the nose Can pass into cells lining respiratory tract Pass through plasma and nuclear membrane Into genome - gets expressed Epithelial cells are replaced every 10-14 days

Question 53

issues with using viruses as a vector for gene therapy

Answer 53

May cause immune response May become immune to virus making deliveries impossible May insert gene in location that disrupts other genes involved in regulating cell division - increases cancer risk Disrupts expression of other genes