Title: Studying whole genomes

1. 04 January 2020 Title: Studying whole genomes Homework: learning package 14 for Thursday

2. Aims from specification • outline the steps involved in sequencing the genome of an organism; (b) outline how gene sequencing allows for genome-wide comparisons between individuals and between species (HSW7b); (c) define the term recombinant DNA;

3. Genomes • 1950’s • Learnt that DNA is the genetic material • Gene technology • Use of DNA to produce something that we want • Developing rapidly • Becoming more and more able to alter genes within organisms • Point to think about • Just because we can do something does that mean that we should do it?

4. Manipulating DNA • Advances in DNA technology • DNA profiling (genetic fingerprinting) • Genomic sequencing • Comparative genome mapping • Genetic engineering • Gene therapy

5. Genome • All the genes possessed by an individual organism, or a population of organisms. • The whole sequence of bases in all of the DNA in an organism.

6. Human Genome Project • 1988 • International project started to discover the sequence of bases in each of the 23 different types of chromosomes found in human cells • 2000 • A working draft sequence was produced

7. Facts about human genome • 99.9% of the base sequence in our DNA seems to be identical in all humans • Variation is caused by the variable 0.1% • This 0.1% is very variable • Variations can be used for DNA profiling • 2% of human genome codes for the manufacture of proteins • Giving around 20 000 genes in the human genome (even mice have more!!) • The rest of the “junk” genome, may be involved in gene expression

8. Chain Termination Method of DNA Sequencing

9. Sequencing a genome • The chain termination method is used to determine the order of bases in a section of DNA • Make multiple labelled copies of each small length of DNA • Lengths of DNA mixed with • DNA Polymerase • Primer • “normal” DNA nucleotides • “labelled” DNA nucleotides • dideoxy nucleotides • Four colours of dye used for bases A, T, G and C • If incorporated in nucleotide chain – chain stops growing

10. Sequencing a genome • Result • Many different chains of different lengths • Each length ends with a labelled nucleotide • Mixture of lengths of DNA separated using electrophoresis • The shorter the length of DNA the faster it travels • Computer records the colours as they pass the end of the tube, if there are enough fragments then every base in the complete chain will be represented. • Computer works out the sequence of the length of DNA

11. Sequencing a genome • Process is largely automated • Put your DNA sample into a sequencing machine • Get a print out from the bottom • Preparation of DNA and analysis is still time consuming

12. Sequencing a genome • Chain-Termination Method can only be used for DNA fragments up to 750bp long • Therefore, the entire genome is broken up and sequenced in sections • Sequencing is carried out on overlapping regions • Stages • Genome mapping • Mechanically break into smaller sections • Carry out sequencing on overlapping sections • Analyse and put back together to form the complete code

13. Whole genome sequencing using BACs

14. 1. Mapping the genome • Genomes are mapped to identify which chromosome or section of chromosome it came from • Uses previous information to help find the location – microsatellites are of use here. • Microsatellites are short sections of DNA (3-4bps) that repeat over thousands of locations on the genome • Samples of genome are mechanically broken into smaller sections ~100,000bps • This is sometimes referred to as the “shotgun” approach.

15. 2. BACs • Sections of DNA placed into bacterial artificial chromosomes (BACs) and transferred to E. Coli cells. • Cells grow in culture, many copies (clones) are produced • These cells are referred to as clone libraries

16. 3. Sequencing BACs • Cells containing specific BACs are taken and cultured. DNA is extracted using restriction enzymes to cut it up into smaller fragments • Different restriction enzymes are used to give different fragment types (will “cut” DNA at different places) • Fragments separated using gel electrophoresis • Fragment sequencing using an automated process • Computer programmes compare overlapping regions from the cuts made by different restriction enzymes in order to reassemble the whole BAC segment sequence.

17. Exam questions • To sequence a small DNA fragment, a single-stranded DNA template and DNA polymerase are needed. • Name the other three reactants needed for a sequencing reaction (3) • Describe and explain the process of sequencing a small DNA fragment (6) 2. The genomes of over 200 different species have been sequenced. Describe how a genome can be sequenced using BACs (8)

18. Answers 1A • DNA primer • Free nucleotides • Fluorescently-labelled modified nucleotides

19. Answers 1B • The reaction mixture is added to four tubes, with a different modified nucleotide in each tube • The tubes undergo PCR to produce lots of strands of DNA of different lengths • Each strand of DNA is a different length because each one terminates at a different point depending on where the modified nucleotide was added • The DNA fragment in each tube are separated by electrophoresis and visualised under UV light • The smallest nucleotide is at the bottom of the gel and each band after this represents one more base added • So the bands can read from the bottom of the gel to the top, forming the base sequence of the DNA fragment

20. Answers 2 • The genome is cut up into smaller fragments using restriction enzymes • The individual fragments are inserted into bacterial artificial chromosomes/BACs, which are inserted into bacteria • Each BAC contains a different DNA fragment, so each bacterium contains a BAC with a different DNA fragment. • The bacteria divide, creating colonies of cloned cells that contain their specific DNA fragment • Together the different colonies make a complete genomic DNA library • DNA is extracted from each colony and cut up using restriction enzymes, producing overlapping pieces of DNA • Each piece of DNA is sequence, using the chain termination method, and the pieces are put back in order to give the full sequence from the BAC • Finally the DNA fragment from each different BAC is put back in order using computers, to compete the entire genome

21. Answers 1B • DNA is mixed with free nucleotides, primers and DNA polymerase • The mixture is heated to 95oC to break the hydrogen bonds • The mixture is then cooled to between 50-65oC to allow the primers to bind/anneal to the DNA • The primers bind/anneal to the DNA because they have a sequence that is complementary to the sequence at the start of the DNA fragment • The mixture is then heated to 72oC and DNA polymerase lines up free nucleotides along each template strand, producing new strands of DNA • The cycle would be repeated over and over to produce lots of copies