Title: Studying whole genomes Homework: learning package 14 for Thursday 21 June 2016

Aims from specification (a)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;

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?

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

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.

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

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 genes in the human genome (even mice have more!!) The rest of the “junk” genome, may be involved in gene expression

Chain Termination Method of DNA Sequencing

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

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

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

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

Whole genome sequencing using BACs

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.

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

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.

Exam questions 1.To sequence a small DNA fragment, a single- stranded DNA template and DNA polymerase are needed. A.Name the other three reactants needed for a sequencing reaction (3) B.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)

Answers 1A 1.DNA primer 2.Free nucleotides 3.Fluorescently-labelled modified nucleotides

Answers 1B 1.The reaction mixture is added to four tubes, with a different modified nucleotide in each tube 2.The tubes undergo PCR to produce lots of strands of DNA of different lengths 3.Each strand of DNA is a different length because each one terminates at a different point depending on where the modified nucleotide was added 4.The DNA fragment in each tube are separated by electrophoresis and visualised under UV light 5.The smallest nucleotide is at the bottom of the gel and each band after this represents one more base added 6.So the bands can read from the bottom of the gel to the top, forming the base sequence of the DNA fragment

Answers 2 1.The genome is cut up into smaller fragments using restriction enzymes 2.The individual fragments are inserted into bacterial artificial chromosomes/BACs, which are inserted into bacteria 3.Each BAC contains a different DNA fragment, so each bacterium contains a BAC with a different DNA fragment. 4.The bacteria divide, creating colonies of cloned cells that contain their specific DNA fragment 5.Together the different colonies make a complete genomic DNA library 6.DNA is extracted from each colony and cut up using restriction enzymes, producing overlapping pieces of DNA 7.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 8.Finally the DNA fragment from each different BAC is put back in order using computers, to compete the entire genome

Answers 1B 1.DNA is mixed with free nucleotides, primers and DNA polymerase 2.The mixture is heated to 95oC to break the hydrogen bonds 3.The mixture is then cooled to between 50-65oC to allow the primers to bind/anneal to the DNA 4.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 5.The mixture is then heated to 72oC and DNA polymerase lines up free nucleotides along each template strand, producing new strands of DNA 6.The cycle would be repeated over and over to produce lots of copies