Genome projects and gene technology

Question 1

what is a genome and what can it include

Answer 1

complete set of genes in a cell. these genes could be arranged on linear DNA, independent chromosomes or on circular DNA in prokaryotes
Can include: gene that codes for a protein
regulatory genes such as those that code for transcription factors
mitochondrial DNA which codes for proteins involved in aerobic respiration, doesn’t have to rely on other cells
chloroplast DNA which codes for proteins involved in protein synthesis
genes that code for antibiotic resistant that are located on plasmids in prokaryotes
viral RNA or DNA such as HIV

Question 2

define phylogenetics

Answer 2

sequence DNA and amino acid sequence helps classify new species

Question 3

what is a proteome and what can it be used to determine

Answer 3

the full range of proteins produced by cells, used to target gene therapy and faulty genes so can make them work.

Question 4

off spec content: Pfizer and BioNTeach

Answer 4

they produce a complementary but shorted mRNA strand to the SARS CoV-2 spike protein. This mRNA strand was the key molecule in the COVID-19 vaccine. When injected into muscle cell in the arm, the mrna was translated into a smaller section of the antigen and this triggered a primary immune response that led to the production of specific antibodies and memory cells that were complementary to part of the antigen.

Question 5

what is recombinant DNA

Answer 5

when a cell has two or more sources of DNA and can be achieved by isolating fragments of the DNA and then inserting this DNA into another organism or species

Question 6

universal

Answer 6

the same triplet codes for the same amino acid, important because the process of transcription and translation is universal meaning that it is possible for a bacterial cell to produce human insulin.

Question 7

degenerative

Answer 7

more than one base triplet for each amino acid

Question 8

non-overlapping

Answer 8

each base is part of only one triplet

Question 9

How can reverse transcriptase be used to isolate a copy of a specific DNA fragment

Answer 9

Reverse transcriptase carries out transcription in reverse. It makes cDNA copies from mRNA templates. mRNA is then mixed with free DNA nucleotides and enzyme reverse transcriptase

Free DNA nucleotides bind to a single stranded mRNA template via complementary base pairing. Reverse transcriptase joins DNA nucleotides together to form a single stranded cDNA molecule. DNA polymerase is required to make cDNA double stranded

Question 10

advantages of using reverse transcriptase

Answer 10

cells contain a maximum of two alleles of each gene (one of each homologous chromosome)
if the cell expressing that gene the cell will contain many mRNA molecules with a complementary base sequence to that gene
mRNA
mRNA is much easier to obtain
using mRNA isolated from cytoplasm means introns have been removed and exons spliced together, whereas gene contains introns and exons
bacterial DNA doesn’t contain introns. Bacteria don’t have enzymes for splicing
DNA fragments produced using mRNA as a template code for functioning protein without the need for splicing
mRNA can be isolated from the cytoplasm of cell types which produce the protein in large amounts

Question 11

how can restriction endonuclease be used to isolate a copy of a specific DNA fragment

Answer 11

these are enzymes that hydrolyse DNA at specific recognition base sequences. Different restriction endonucleases hydrolyse at different points because the shape of the sequence is complementary to the enzymes active site.

DNA is incubated with the specific enzyme which hydrolyses DNA into fragments wherever the recognition sequence appears. if target gene has recognition sequences before and after the target gene, the fragment will contain the desired gene.

If the recognition sequence for the selected restriction endonuclease occurs within the DNA fragment, you want to isolate it.- this will cut the gene and it will not code for a functional protein

Question 12

What are recognition sequences

Answer 12

palindromic, reads the same both forwards and backwards

Question 13

How does the gene machine work

Answer 13

way of manufacturing genes by entering the desired sequence of nucleotide bases into a computer
can also reverse engineer the nucleotide sequence by working out the primary amino acid structure

1. single stranded DNA produced
   2.PCR machine: ssDNA copied and made double stranded (dsDNA) of the gene of interest
2. PCR amplifies the target gene

Question 14

describe the process of the gene machine

Answer 14

desired nucleotide fed into computer, synthesis of short sequence of nucleotides, assembly of gene, these short sequences are overlapped then joined together and made double stranded using the polymerase chain reaction, gene is inserted into bacterial plasmid

Question 15

advantages of isolating DNA fragments via the gene machine

Answer 15

artificial genes are easily transcribed and translated by prokaryotes, as they have no introns in their DNA.

Question 16

define a vector

Answer 16

a DNA carrier used to transfer foreign DNA into cells

Question 17

describe the process of inserting genes into a vector

Answer 17

isolated target DNA fragments inserted into the vector DNA by cutting open the vector using the same restriction endonuclease that was used to isolate the DNA fragment. Use the same to create complementary base pairs

this produces complementary sticky ends between the ends of DNA fragment and cut ends of vector DNA

Target DNA fragment anneals to vector DNA by complementary base pairing between their sticky ends

DNA ligase is used to join the DNA fragment and vector DNA at the sugar phosphate backbone. This process is called ligation and forms phosphodiester bonds.

The new recombined DNA fragment and vector DNA is recombinant DNA.

Question 18

outline a method for in vivo gene cloning

Answer 18

cut desired gene from the DNA of the desired organism
using restriction endonuclease
make artificial DNA with correct sequence of bases
using DNA polymerase
cut plasmid open
with same restriction endonuclease
sticky ends attach
DNA ligase used to join and return plasmid to bacterial cells

Question 19

why must organisms be identified

Answer 19

not all vectors take up target gene to become recombinant
not all host cells become transformed, by taking up recombinant vectors
Only transformed host cells will contain the target gene and will synthesise desired protein, scientists only want to clone the transformed host cells

Question 20

define marker gene

Answer 20

allows easy identification of cells that have taken up genetically transformed plasmid

Question 21

name the common types of marker genes

Answer 21

antibiotic resistant gene- marker gene codes for antibiotic resistance, when cells are grown in the presence of this antibiotic only transformed organisms will grow

fluorescence gene- marker gene codes for a protein that fluoresces when exposed to UV light. Identified transformed organisms are cultured to produce large genetically identical populations

enzyme- another gene marker is the gene that codes for the synthesis of the enzyme lactase which will turn colorless substrate blue

Question 22

identifying transformed bacteria using antibiotic resistant genes

Answer 22

some cells will not have taken up any plasmid at all. These cells are killed by both types of antibiotic
some cells will have taken up transformed plasmid. these cells are resistant to one type of antibiotic, but not the second
some cells will have taken up original plasmid, these are resistant to one type of antibiotic

Question 23

cloning in vitro via PCR

Answer 23

polymerase chain reaction is used to amplify

the stages are: Separation- heat to 95 to separate the weak hydrogen bonds between complementary base pairs.
annealing- cool to 55 and add DNA primers which will attach to complementary base pairs and allow DNA polymerase to bind
Synthesis- heat to 72 so thermo-stable DNA polymerase can bind allowing the synthesis of nucleotides and the formation of phosphodiester bond.

Question 24

define primer

Answer 24

short pieces of single stranded DNA with complementary base sequences to bases at the start of DNA fragment you want to isolate. primers also prevent DNA strands from sticking back together and allow DNA polymerase to attach and join free nucleotides together

Question 25

how to calculate the number of DNA strands you have after a number of cycles

Answer 25

2^n

Question 26

how to calculate the number of cycles from the number of DNA strands you used

Answer 26

log2(number of DNA molecules)

Question 27

describe the process of PCR

Answer 27

heat DNA to 95 which breaks weak hydrogen bonds
add primers and nucleotides
cool to 50 to allow binding of nucleotides and primers
add thermo-stable DNA polymerase
heat to 75
DNA polymerase joins nucleotides together
repeat cycles many times

Question 28

in vitro vs in vivo DNA fragment cloning

Answer 28

In Vivo
Can be used to produce protein or mRNA from the inserted DNA as well as the target DNA
Cells have mechanisms for correcting errors that are made when copying the gene, almost no risk of contamination as they are pure samples
can be used to clone large DNA fragments
slower-one DNA replication per cell division, limited by the growth of host cell
DNA,RNA and proteins produced are modified
need to isolate DNA fragment from DNA before it can be inserted into the host cell
can’t be used to copy partly broken down DNA
not very sensitive- need large DNA sample to begin with

In vitro
can only be used to copy DNA
lacks error-correcting mechanisms, so the error rate is high
cloning becomes unreliable when used to copy DNA fragments longer than 1000 base pairs
quicker
no modification of DNA
only replicates target DNA, no need to isolate DNA fragment
can be used to copy partly broken down DNA
very sensitive- only requires a small DNA sample to start with

Question 29

benefits of recombinant DNA technology

Answer 29

develop medical applications- to produce faulty or lack of protein
develop agricultural applications- produce crops with larger yield etc
better understanding of biological processes, including DNA replication, mitosis and cell death
design new or improve existing industrial processes, produce large quantities of enzymes quickly and cheaply

Question 30

concerns regarding recombinant DNA technology

Answer 30

antibiotic resistance may spread
inserting new genes into a crop plant may disrupt other genes creating toxic products within genetically modified food
introducing herbicide resistance genes to crop plants may transfer to wild species producing super weeds
technology may become concentrated in the hands of large corporations

Question 31

using viruses as vectors

Answer 31

If foreign DNA fragment is introduced into the viral genetic material, it will insert the foreign gene at the same time as its own genetic material. Viral DNA is cut using the same restriction endonuclease and joined to foreign DNA
viruses can cause an immune response and the formation of cytotoxic killer cells and memory B cells. To combat this, vector viruses are further modified to evade immune response.

Question 32

Cystic fibrosis

Answer 32

caused by a mutation in the gene that codes for cytic fibrosis transmembrane consuctance regulator protein. This gene is expressed mainly in the cells that line the airways producing thick music that leads to infection,

Question 33

why are viruses used as vectors

Answer 33

as plasmids cannot be carry DNA into human cells

Question 34

somatic gene therapy

Answer 34

DNA transfer to our normal body tissue- non hereditable

Question 34

why are bacteria used

Answer 34

because they are fast replicating

Question 34

germ line gene therapy

Answer 34

DNA trasnfers to cells that produce eggs or sperm so is hereditable

Question 34

How many base sequences long are DNA probes

Answer 34

10

Question 35

What is DNA hybridisation

Answer 35

When the DNA probe binds to the target DNA, it is now half fluorescent probe and half target DNA isolated from the patient

Question 35

What can the prescience of fluorescence allow genetic counsellors to conclude

Answer 35

That the individual carries a recessive allele known to cause a genetic disorder
That the individual contains mutations in known genes linked to increased risk of disease
Contains genes that code for proteins that predict positive or negative response to a drug dosage

Question 35

Gene probe definition

Answer 35

A short, single stranded DNA molecule with complementary base sequence to DNA fragments to be located which is radioactive or labelled by a fluorescent molecule.

Question 35

What does DNA fingerprinting use and what does VNTR stand for

Answer 35

Uses the region of DNA between genes, Variable number tandem repeats- found on the chromosome and are non-coding sections of DNA

Question 36

Limitations of somatic gene therapy

Answer 36

Not all cells take up new DNA
Not all cells express DNA allele
Only some tissue types are accessible
Multiple treatments may be needed
Body can produce immune response to vector

Question 37

Describe genetic screening using DNA probes and DNA hybridisation

Answer 37

The sequence of nucleotides on the mutated gene is determined by DNA base sequencing. Genetic libraries now store the DNA sequences of many of the genes responsible for genetic diseases
Fragment of dna with complementary bases to the mutant allele of the gene is produced
DNA probe is formed by flourescently labelling the dna fragments
Pcr technique are used to produce multiple copies of the DNA probe
Probe is added to single stranded dna fragments from the person being screened
If the donor has mutated gene, some donor DNA fragments will have a base sequence that is complementary to the probe will bind to its complementary bases on the donor DNA.
These DNA fragments will now be labelled with the probe and can be distinguished from the rest of the fragments
If complementary fragments are present, the DNA probe will be taken up and the dye will fluoresce- detected by a special microscope.

Question 38

How can the different length of VNRTs be analysed

Answer 38

Gel electrophoresis

Question 39

Describe how genetic finger printing is carried out

Answer 39

DNA extracted from sample
DNA cut/hydrolysed into segments using restriction endonuclease
Must leave VNRTR mini satellites intact
DNA fragments separated using electrophoresis
Mixture put into wells on gel and electric current passed through
Immerse gel in alkaline solution so two strands of DNA are separated
Radioactive marker/probe added
Areas with probe identified using x-ray film/autoradiography

Question 40

How to tell if someone is the father or committed a crime?

Answer 40

Bands in sample must be the same