6.1.3 - Manipulating Genomes (set A - DNA Profiles + DNA Sequencing)

Question 1

Define genome?

Answer 1

Genome of an organism is all the genetic material it contains

Question 2

What are introns?

Answer 2

Large non-coding regions of DNA that are removed from mRNA before its translated into a polypeptide chain

• within introns there are short sequences of DNA repeated many times - known as satellite DNA

Question 3

Explain what minisatellite and microsatellite DNA are?

Answer 3

both types of highly repetitive DNA that are crucial for maintaining the structure of the genome. They are both non-coding DNA within introns, telomeres and centromeres

Microsatellites are short tandem repeats (STRs) that are typically 1–6 base pairs long, but can sometimes be up to 10 base pairs

Mini-satellites are longer, with repeats of 10–100 base pairs

Question 4

Explain the function and basis of STRs in DNA profiling?

Answer 4

STRs represent repetitive regions of the genome, which have sequences made up of repeating units of nucleotides - vary between individuals

• satellites always occur in the same pace on chromosomes - but vary in length (only identical twins have identical satellites)

used for DNA profiling

Question 5

Outline the 5 main stages of producing a DNA profile?

Answer 5

1) extraction of DNA - technique of PCR allows tiny fragments of tissue to be used to give enough of needed DNA

2) digesting the sample - restriction endonuclease enzymes cut strands of DNA into fragments - different restriction endonuclease cut DNA at restriction site

3) separating DNA fragments - electrophoresis used - single-stranded DNA fragments transferred onto a membrane by southern blotting

4) hybridisation - fluorescent DNA probes added - identify the microsatellite regions (excess probes washed off)

5) Seeing evidence - labels were added to probes and correct technique then used (eg paper placed under UV light) - give pattern of bars (DNA profile)

Question 6

Explain in depth how the sample is digested when producing a DNA profile?

Answer 6

• restriction endonuclease enzymes cut strands into small fragments
• other restriction endonucleases cut DNA at a specific nucleotide sequence (restriction site)

restriction endonucleases give ability to cut DNA strands at defined points in introns - mixture of different ones which leave the repeating units (satellites) intact

Question 7

Outline the role of restriction endonuclease?

Answer 7

• enzyme
• used in DNA profiling to cut DNA into small fragments and again to cut DNA at a specific nucleotide sequence
• make 2 cuts - one through each strand of DNA double helix

cut the 2 DNA strands unevenly - leaving one of the stand of the DNA fragment a few bases loner than the other strand (sticky ends) easier to insert the desired gene into the DNA of a different organism

Question 8

Explain in depth how the sample is separated when producing a DNA profile - mention electrophoresis?

Answer 8

Technique which utilises way charged particles move through a gel medium, under influence of an electric current

• gel immersed in alkali (in order to separate DNA double strands into single) - single-stranded DNA fragments than transferred onto a membrane by southern blotting

Question 9

Explain in depth the role of hybridisation when producing a DNA profile?

Answer 9

• radioactive or fluorescent DNA probes added
• DNA probes are short DNA or RNA sequences complementary to a known DNA sequence - bind to complementary strand of DNA under particular conditions of pH and temperature
• probes identify microsatellite regions (which are more varied than larger minisatellite)

Question 10

Explain in depth the how the DNA profile is analysed to finally create a profile at the final step?

Answer 10

• if radioactive labels added to proves - x-ray images are taken of the paper/membrane
• if fluorescent label added to probes - paper/membrane placed under UV light so the tags glow (main method)

fragments give pattern of bars (DNA profile) - unique to every individual expect identical siblings

Question 11

Outline the steps of the process of the polymerase chain reaction (PCR)?

Answer 11

1) separating double-stranded DNA sample - temperature in PCR machine set to 90 degrees for 30 seconds (denatures DNA by breaking hydrogen bonds - separation the strands)

2) add primers and reduce temp to 55 degrees (allows primers to anneal)

3) synthesis of DNA - temperature is increased to 72-75 degrees for 1 min (optimum temp for DNA polymerase) DNA polymerase adds bases (nucleotides) to primer (building up complementary strands of DNA - which are double-stranded and identical to original sequence)

Question 12

Explain the point of polymerase chain reaction (PCR)?

Answer 12

DNA profiling often used to solve crimes

• only very tiny amounts of DNA available - PCR allows scientists to produce a lot of DNA from a tiny sample

Question 13

Outline 4 use of DNA profiling?

Answer 13

• criminal investigations - DNA profile compared to sample from suspect or to criminal database
• prove paternity of a child
• identifying species to which an organism belongs
• identifying individuals at risk of developing particular diseases

Question 14

Define DNA sequencing?

Answer 14

Process of determining the precise order of nucleotides within a DNA molecule

Question 15

Explain early research into DNA sequencing - reference Sanger sequencing?

Answer 15

Team of scientists developed early technique which involved radioactive labelling of bases and gel electrophoresis - carried out manually (took a long time)

• new technique Sanger sequencing developed which allowed sequences of 500-800 bases to be read at a time (later improved with switching to coloured fluorescent tags) used in the human genome project (HGP)

Question 16

explain the human genome project (HGP)?

Answer 16

• massive international project - where scientists worked together to map the entire human genome
• development and automation of sequencing techniques and computers meant the 15 year goal was completed earlier

Question 17

Outline the process of DNA sequencing?

Answer 17

1) DNA mixed with primer (DNA polymerase + excess of nucleotides A,T,C,G and terminator bases)

2) mixture placed in thermal cycler - changes temp separating double stranded DNA at 96 degrees and allowing primers to annal to DNA strand at 50

3) at 60 degrees polymerase builds up new DNA strand by adding nucleotides with complementary bases to single strand template

4) synthesis of DNA terminated each time a terminator base is incorporated - results in many DNA fragments at different lengths (fragments separated by length by capillary sequencing)

5) fluorescent markers on terminator bases used to identify the final base on each fragment - lasers detect them and thus the order of the sequence - order of bases in capillary tubes show sequence of new, complementary strand of DNA (used to build up sequence of original DNA)

Question 18

Explain what terminator bases are?

Answer 18

• used in DNA sequencing
• modified versions of 4 nucleotide bases A,T,C and G - which stop DNA synthesis when they are included
• terminator bases given coloured fluorescent tags

Question 19

Explain what ‘massively parallel sequencing is’ and how it works?

Answer 19

• sequencing reaction takes place on plastic slide (called a flow cell)
• millions of fragments of DNA are attached to the slide and replicated using PCR to form clusters of identical DNA fragments
• sequencing process uses coloured terminator bases to stop the reaction so an image can be taken
• clusters sequenced at same time - much faster

Question 20

Define bioinformatics?

Answer 20

Development of software and computing tools needed to organise and analyse raw biological data (includes development of algorithms, mathematical models and statistical tests)

Question 21

Define computational biology?

Answer 21

Use of data to build theoretical models of biological systems - can be used to predict what will happen in different circumstances

Question 22

Outline the implications of analysing the human genome?

Answer 22

• computers can analyse and compare the genomes of many individuals - reveals pattern of DNA and diseases we are vulnerable
• implications for health management and field of medicine

Question 23

Outline and explain 3 points the analyse of pathogens enables?

Answer 23

• allows doctors to find out source of an infection
• to identify antibiotic-resistant strands of bacteria - ensuring antibiotics are only used when they will be effective
• allows scientists to identify regions in the genome of pathogen - may be useful targets for developing new drugs

Question 24

Give 2 other uses of DNA sequencing?

Answer 24

• identifying species - by comparing particular sections of the genome common to all species but vary between them
• searching for evolutionary relationships - DNA sequences of different organisms can be compared and basic mutation rate can be calculated - scientists can work out how long ago 2 species diverged from a common ancestor

Question 25

Explain what proteomics is?

Answer 25

Study of amino acid sequencing of an organisms entire protein complement

• traditionally believed each gene codes for specific protein - however some genes can code for many different proteins
• around 20-25,000 coding genes in human DNA but estimates of around 250,000 to 1,000,000 different proteins

Question 26

Define synthetic biology?

Answer 26

An emerging area of research that can be described as the design and construction of novel artificial biological pathways, organisms or devices or the redesign of existing natural biological systems

Question 27

Give and explain 3 different techniques of synthetic biology?

Answer 27

• genetic engineering - may involve a single or major genetic modification of an organism
• use of biological systems in industrial contexts - eg production of drugs from microorganisms
• synthesis of new genes to replace faulty genes - eg in develop treatments for cystic fibrosis (CF)