biology module 5 manipulating genomes

what is DNA sequencing

allows the nucleotide base sequence of an organisms genetic material to be identified

who developed the method of chain sequencing and what is this method known as

Frederick Sanger. Sanger sequencing

what are dideoxynucleotides

used in sanger sequencing. modified nucleotides which can pair with nucleotides on the template strand if they have a complementary base

why is it known as chain termination

because when DNA polymerase encounters a dideoxynucleotide it stops replicating

step 1 of chain termination

4 test tubes prepared, contains DNA sample, DNA polymerase, primers, nucleotides, and one of the four types of dideoxynucleotides

step 2 of chain termination

test tubes are incubated and the primer anneals (sticks) to the sample, producing double stranded DNA at the start of the sequence

step 3 of chain termination

DNA polymerase attaches to this double stranded section and begins DNA replication using the free nucleotides. Hydrogen bonds form between the complementary bases on the nucleotides

step 4 of chain termination

at any time, DNA polymerase can insert one of the dideoxynucleotides by chance which results in the termination of replication

step 5 of chain termination

complementary DNA chains of varying lengths are produced because the point at which the dideoxynucleotide is inserted varies with every strand

step 6 of chain termination

once the incubation period has ended, the new DNA chains are separated from the template DNA

step 7 of chain termination

the resulting single stranded DNA chains separated according to length using gel electrophoresis. the gel will have four wells, one each for the dideoxynucleotides

what is a genome

contains all the genes within an organism

how can you determine protein sequences from the genetic code

the genetic code can be used to predict the amino acid sequence within a protein. can predict how the new protein will fold into its tertiary structure

what is bioinformatics

a field of biology which involves the storage, retrieval, and analysis of data from biological studies. once a genome is sequenced, bioinformatics allows scientists to make comparisons with the genomes of other organisms

how can genetic variation be investigated

the genetic variation within a species can be investigated as many individuals of the same species can have their genomes compared. if there’s a large number of differences then there is higher genetic variation

how can evolutionary relationships be investigated

by comparing the genomes of different species. species with a small number of differences between their genomes share a more recent common ancestor

what is epdimiology

study of the spread of infectious disease within populations. the genomes of pathogens can be sequenced and analysed to aid research

the human genome project

1990, DNA samples taken from multiple people around the world and sequenced to create a reference genome

why is determining the proteome of humans difficult

large amounts of non coding DNA are present in human genomes and it can be very hard to identify these sections of DNA from the coding DNA

why is the proteome larger than the genome

due to alternative splicing and post-translational modification of proteins

what does alternative splicing allow for

a single gene to produce multiple proteins

what is synthetic biology

a recent area of research that aims to create new biological parts, devices and systems. involves large alterations to an organisms genome

what does PCR stand for

polymerase chain reaction

what is PCR

a common molecular biology technique used in most applications of gene biology. used to produce large quantities of specific fragments of DNA or RNA from very small quantities

requirements for PCR

1. target dna or rna being amplified

2. primers

3. DNA polymerase

4. free nucleotides

   1. buffer solution

what is a primer

short sequences of single stranded DNA that have sequences complementary to the end of DNA or RNA

what is the DNA polymerase used for in PCR

enzyme used to build the new DNA/RNA strand

what are the free nucleotides used for in PCR

used in construction of DNA/RNA strands

what is the buffer solution used for in PCR

to provide the optimum pH

how many molecules of DNA are produced in each cycle of PCR

doubles every cycle. in a standard round of 20 cycles. approximately a million DNA molecules are produced

which piece of equipment do you need for PCR

occurs in a thermocycler which automatically provides the optimal temperature for each stage

describe the stage of denaturation for PCR

double stranded DNA is heated to 95 degrees celcius which breaks the hydrogen bonds that bond the two DNA strands together

describe the stage of annealing

temperature is decreased to 50-60 degrees so the primers can anneal to the ends of the single strands of DNA

describe the stage of elongation/extension for PCR

temperature increased to 72 as this is the optimum temperature for polymerase to build the complementary DNA strands to produce the new identical double stranded DNA molecules

what is gel electrophoresis

a technique used widely in the analysis of DNA, RNA, and proteins. molecules seperated according to their size/mass and their net charge

why are they separated according to charge

positively charged molecules will move towards the negative pole cathode) while negatively charged molecules move towards the positive pole (anode). DNA is negatively charged so will move towards the anode

why are they separated according to size/mass

different sized molecules move through the gel at different rates. smaller molecules move faster than large molecules

how do you prepare the DNA for gel electrophoresis

you need to amplify the number of fragments using PCR. then restriction endonucleases are used to cut the DNA into fragments. scientists use enzymes that will cut close to the variable number tandem repeat regions

what are variable number tandem repeat regions

regions found in the non-coding part of DNA. contain variable numbers of repeated DNA sequences and are known to vary between different people

step 1 of gel electrophoresis

create an agarose gel plate in a tank. wells are cut into the gel at one end

step 2 of gel electrophoresis

submerge the gel in an electrolyte solution in the tank

step 3 of gel electrophoresis

load the fragments into the wells using a micro pipette

step 4 of gel electrophoresis

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 due to the attraction between the negative charged phosphates of DNA and the anode

step 5 of gel electrophoresis

the smaller mass of DNA fragments will move faster and further from the wells than the larger fragments

step 6 of gel electrophoresis

the fragments are not visible so must be transferred onto absorbent paper or nitrocellulose which is then heated to separate the two DNA 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

what are probes

single stranded DNA sequences that are complementary to the VNTR regions. probes can be identified using a radioactive label or a fluorescent stain

what can DNA profiling be used for

identifying suspects for a crime and identify corpses. because everyone has repeating short non-coding regions of DNA that are unique to them

how do you create a DNA profile

1. obtain the DNA

2. increase the quantity using PCR

3. use restriction endonucleases to cut the amplified DNA molecules into fragments

4. separate fragments using gel electrophoresis

5. add probes that will bind to specific VNTR regions

6. X-ray images are produced or UV light is used to create images of the fluorescent labels glowing. these images contain patterns of bars which are then analysed

what is genetic engineering

the manipulation of the DNA sequences of an organism

how can you artificially change an organisms DNA

by combining lengths of nucleotides from different sources

what is recombinant DNA

altered DNA with the introduced nucleotides

what is a transgenic organism

an organism which contains nucleotide sequences from a different species

what is a GMO

a genetically modified organism - am organism that has introduced genetic material

uses of genetic engineering

genetic modification of crops to increase crop yield.

genetic modification of livestock to give disease and pest resistance

genetic modification of bacteria to produce medicines

what steps must be taken for an organism to be genetically engineered

1. identification of the DNA fragment or gene

2. isolation of the desired DNA fragment

3. multiplication of the DNA fragment

4. transfer into the organism using a vector

   1. identification of the cells within the new DNA fragment

the use of enzymes in genetic engineering

-restriction endonucleases used to cut genes at specific base sequences

-ligase used to join together the cut ends of DNA by forming phosphodiester bonds

-reverse transcriptase used to build double stranded DNA from single stranded RNA

the use of vectors in genetic engineering

-used to deliver DNA fragments into a cell

-plasmids used to transfer DNA into bacteria or yeast

-viruses used to transfer DNA into human cells or bacteria

-liposomes fuse with cell membranes to transfer DNA into cells

the use of markers in genetic engineering

-genes that code for identifiable substances that can be tracked

-fluorescent markers fluoresces under UV light

-enzyme markers transforms colourless substrates into products that are coloured

-antibiotic resistance marker genes are inserted into a gene for antibiotic resistance. this activates the antibiotic resistance gene and means that successfully transformed bacteria will be wiped out if exposed to the antibiotic

what is gene therapy

using various mechanisms to alter a persons genetic material to treat or cure diseases

why are most gene therapies still in the clinic

because scientists are having difficulty finding delivery systems that can transfer normal alleles into a persons cells and how to ensure the gene is correctly expressed once there

what are the most common vectors used in gene therapy

viruses because they have the mechanisms needed to recognise cells and deliver the genetic material into them

what are the two types of somatic gene therapy

1. in vivo

2. ex vivo

why is germ cell gene therapy illegal in humans

any changes made to the genetic material of these cells is potentially permanent and could be inherited by future generations