Manipulating Genomes 6.3

Question 1

What is DNA sequencing?

Answer 1

-a technique that allows genes to be isolated and read

Question 2

How has DNA sequencing developed?

Answer 2

-the techniques used in sequencing have rapidly advanced from Sanger sequencing to high throughput sequencing

Question 3

What is high throughput sequencing?

Answer 3

-when many fragments are processed and sequenced simultaneously
-this has made DNA sequencing more efficient

Question 4

What are the principles of DNA sequencing?

Answer 4

• The DNA sample is divided into four separate sequencing reactions which contain all four standard nucleotides, DNA polymerase, primers required for replication and modified nucleotides which have been fluorescently labelled for ease of identification.
• When a modified nucleotide is incorporated into a growing chain, replication is terminated
• DNA fragments of different lengths are formed across the reaction vessels
• High resolution electrophoresis is used to separate the fragments by size - single base differences can be seen
• The fragments are visualised under UV light, thus enabling the base sequence to be read from the bottom of the gel upwards

Question 5

Gene sequencing has allowed for _________ comparisons between individuals and between _____.

Answer 5

-genome-wide
-species

Question 6

How has gene sequencing allowed for the sequences of amino acids in polypeptides to be predicted?

Answer 6

-the genetic code can be used to predict the amino acid sequence within a protein
-once scientists know the amino acid sequence they can predict how the new protein will fold into its tertiary structure
-the researchers need to know which part of the gene codes for exons and which codes for introns
-this information can be used for a range of applications such as in synthetic biology

Question 7

What is Bioinformatics?

Answer 7

-field of biology that involves the storage, retrieval and analysis of data from biological studies
-These studies may generate data on DNA sequences, RNA sequences, and protein sequences, as well as on the relationship between genotype and phenotype
-High-power computers are required to create databases
-The large databases contain information about an organism’s gene sequences and amino acid/protein sequences
-Once a genome is sequenced, bioinformatics allows scientists to make comparisons with the genomes of other organisms using the many databases available

Question 8

What has bioinformatics and computational biology contributed to?

Answer 8

There are contributing to biological research into:

-genotype-phenotype relationships
-epidemiology
-searching for evolutionary relationships

Question 9

What are two uses of DNA profiling ?

Answer 9

-forensics
-analysis of disease risk

Question 10

How is DNA profiling carried out?

Answer 10

1-DNA is obtained from the individual (either by mouth swab, from blood, hair, from bone in ancient remains)
2-the DNA is then digested with restriction enzymes. These enzymes cut the DNA at specific recognition sites. They will cut it into fragments which will vary in size from individual to individual
3-the fragments are separated by gel electrophoresis and stained. Larger fragments travel the shortest distance in the gel
4- a banding pattern can be seen
5-the DNA to which the individual’s is being compared is treated with the same restriction enzymes and also subject to electrophoresis
6- the banding patterns of the DNA samples can then be compared

Question 11

What is polymerase chain reaction (PCR) ?

Answer 11

-a biomedical technology in molecular biology that can amplify a short length of DNA to make a large number of copies

Question 12

What facts does PCR rely on?

Answer 12

-that DNA is made of 2 anti parallel backbone strands
-each strand of DNA has a 5’ end and a 3’ end
-base pairs pair up according to complementary base pairing rules A with T and G with C

Question 13

How does PCR differ from DNA replication?

Answer 13

-only short sequences of DNA can be replicated not entire chromosomes
-it requires the addition of primer molecules to make the process start
-a cycle of heating and cooling is needed to separate the DNA strands, bind primers to the strands and for the DNA strands to be replicated

Question 14

What are the 3 key stages of PCR?

Answer 14

The PCR process occurs in a piece of specialist equipment called a thermal cycler, which automatically provides the optimal temperature for each stage and controls the length of time spent at each stage
The three stages are:
-Denaturation – the double-stranded DNA is heated to 95°C which breaks the hydrogen bonds that bond the two DNA strands together
-Annealing – the temperature is decreased to between 50 - 60°C so that primers (forward and reverse ones) can anneal to the ends of the single strands of DNA
-Elongation / Extension – the temperature is increased to 72°C for at least a minute, as this is the optimum temperature for Taq polymerase to build the complementary strands of DNA to produce the new identical double-stranded DNA molecules

This process is repeated for many cycles. In each cycle the DNA is doubled

Question 15

Why is Taq DNA polymerase used in PCR?

Answer 15

-as it comes from thermophilic bacteria (bacteria that live at high temperatures)
-this means it does not denature at the high temperatures involved during the first stage of the PCR reaction
-as well as this its optimum temperature is high enough to prevent annealing of the DNA strands that have not been copied yet

Question 16

What does a buffer solution do?

Answer 16

Provides the optimum pH for the reactions to occur in

Question 17

What are primers in PCR?

Answer 17

Primers (forward and reverse) – these are short sequences of single-stranded DNA that have base sequences complementary to the 3’ end of the DNA or RNA being copied. They define the region that is to be amplified by identifying to the DNA polymerase where to begin building the new strands

Question 18

What are some applications of PCR?

Answer 18

-tissue typing ->donor and recipient tissues can be typed prior to implantation to reduce the risk of rejection of the transplant
-detecting mutations-> DNA can be analysed for the presence of a mutation that leads to a genetic disease
-identifying viral infections
-monitoring the spread of infectious disease
-forensic science

Question 19

What is electrophoresis ?

Answer 19

Process used to separate the DNA fragments and proteins according to their size using an electric current

Question 20

Why does electrophoresis work?

Answer 20

This separation occurs because:
-The electrical charge molecules carry:
Positively charged molecules will move towards the cathode (negative pole) whereas negatively charged molecules will move towards the anode (positive pole) eg. DNA is negatively charged due to the phosphate groups and thus when placed in an electric field the molecules move towards the anode
-The different sizes of the molecules:
Different sized molecules move through the gel (agarose for DNA and polyacrylamide – for proteins) at different rates. The tiny pores in the gel result in smaller molecules moving quickly, whereas larger molecules move slowly
The type of gel:
Different gels have different sized pores which affect the speed at which the molecules can move through them

Question 21

How is DNA prepared for electrophoresis?

Answer 21

-scientists increase (amplify) the number of DNA molecules by the polymerase chain reaction (PCR).
-Then restriction endonucleases (enzymes) are used to cut the DNA into fragments
-Different restriction enzymes cut the DNA at different base sequences. Therefore scientists use enzymes that will cut close to the variable number tandem repeat (VNTR) regions
-Variable number tandem repeats (VNTRs) are regions found in the non-coding part of DNA. They contain variable numbers of repeated DNA sequences and are known to vary between different people. These VNTR may be referred to as ‘satellite’ or ‘microsatellite’ DNA

Question 22

What is the method for gel electrophoresis?

Answer 22

1-Create an agarose gel plate in a tank. Wells (a series of groves) are cut into the gel at one end
2-Submerge the gel in an electrolyte solution (a salt solution that conducts electricity) in the tank
3-Load (insert) the fragments into the wells using a micropipette
4-Apply an electrical current to the tank. The negative electrode must be connected to the end of the plate with the wells as the DNA fragments will then move towards the anode (positive pole) due to the attraction between the negatively charged phosphates of DNA and the anode
5-The smaller mass / shorter pieces of DNA fragments will move faster and further from the wells than the larger fragments
6-The fragments are not visible so must be transferred onto absorbent paper or nitrocellulose which is then heated to separate the two DNA strands. Probes are then added, after which an X-ray image is taken or UV-light is shone onto the paper producing a pattern of bands which is generally compared to a control fragment of DNA

Question 23

What are probes in electrophoresis

Answer 23

Probes are single-stranded DNA sequences that are complementary to the VNTR regions sought by the scientists. The probes also contain a means by which to be identified. This can either be:
-A radioactive label (eg. a phosphorus isotope) which causes the probes to emit radiation that makes the X-ray film go dark, creating a pattern of dark bands
-A fluorescent stain / dye (eg. ethidium bromide) which fluoresces (shines) when exposed to ultraviolet (UV) light, creating a pattern of coloured bands

Question 24

Why are probes useful?

Answer 24

They are useful in locating specific DNA sequences for example:
-to locate a specific gene needed for use in genetic engineering
-to identify the same gene in a variety of different genomes from different species when conducting genome comparison studies
-to identify the presence or absence of a specific allele for a particular genetic disease or that gives susceptibility to a particular condition

Question 25

How do you separate proteins for electrophoresis?

Answer 25

The different amino acids (because of the different R groups) determine the charge of proteins. The charge of the R groups depends on the pH and therefore buffer solutions are used during the separation of proteins to keep the pH constant
Proteins are prepared for electrophoresis by:
-Denaturing (to break the disulfide bonds)
-Then manipulating the proteins into rod shapes (which are negatively charged) to allow separation by size

Question 26

What can separating proteins by electrophoresis be used for?

Answer 26

Gel electrophoresis can be used to show genotypes of individuals by separating polypeptide chains produced by different alleles
eg. The haemoglobin variants, α-globin, β-globin and the sickle cell anaemia variant of β-globin, have different net charges and therefore will separate out during electrophoresis to show the presence of the sickle cell allele

Question 27

What stages are necessary in genetic engineering?

Answer 27

-Identification of the desired DNA fragment or gene
-Isolation of the desired DNA fragment (restriction enzymes are used to create sticky ends)
-Multiplication of the DNA fragment (using polymerase chain reaction - PCR)
-Transfer desired gene into the organism using a vector. This is (e.g. plasmids, viruses, liposomes).
-Electroporation is used to encourage uptake of plasmid vectors. By increasing the permeability of bacterial membranes via the use of calcium salts and rapid temperature change from O to 40 degrees.
-Identification of the cells with the new DNA fragment (by using a marker), which is then cloned

Question 28

What are the ethical issues relating to the genetic manipulation of animals (including humans), plants and microorganisms?
Include both the positive and negative

Answer 28

There are many ethical considerations regarding genetic engineering. Benefits of genetic engineering include insect resistance in crops such as soya and genetically used animals used to produce pharmaceuticals. Some people object to genetic engineering due to the potential effect it might have on the environment, or because of the idea that genetically modified seeds would not be as easily available to poorer farmers.

Question 29

What are the principles of gene therapy?

Answer 29

-the main principle of gene therapy is to insert a functional allele of a particular gene into cells that contain only mutated and non functioning alleles of that gene
-if the inserted allele is expressed then the individual will produce a functioning protein and no longer have the symptoms associated with the genetic disorder

Question 30

What is somatic cell gene therapy?

Answer 30

-gene therapy by inserting functional alleles into body cells
-affects only certain cells
-the alterations made to the patient’s genome are not passed to the patients offspring

Question 31

What is germ line gene therapy?

Answer 31

-gene therapy by inserting functional alleles into gametes or zygotes
-all the cells of that individual will be altered and their offspring may also inherit the foreign allele

Question 32

Describe the process of genetic engineering

Answer 32

1-obtaining the required gene- mRNA can be obtained from the cells where the gene is being expressed an enzyme called reverse transcriptase can then catalyse the formation of a single strand of complementary DNA (cDNA) using the mRNA as a template. The addition of primers and DNA polymerase can make this cDNA into a double strand length of DNA whose base sequence codes for the original protein or a DNA probe can be used to locate a gene within the genome and restriction enzymes can be used to cut it out
2-placing the gene into a vector- plasmids can be obtained from organisms such as bacteria and mixed with restriction enzymes that will cut the plasmid at specific recognition sites. The cut plasmid has exposed unpaired nucleotide bases called sticky ends. If free nucleotide bases complementary to the sticky ends of the plasmid are added to the ends of the gene to be inserted then the gene and the cut plasmid should anneal. DNA ligase enzymes catalyses the annealing to form recombinant DNA
3- getting the vector into the recipient cell- electroporation is used - a high voltage pulse is applied to the cell to disrupt the membrane