DNA SEQUENCING ASSIGNMENT

In this assignment you will be using the Sanger (dideoxy) method to sequence a part of the human insulin gene. The procedure involves setting up four reactions. Each reaction contains the human insulin DNA sample, an excess of the four deoxynucleotides (dA, dG, dC and dT), DNA polymerase and a limiting amount of one of the four dideoxynucleotides (ddA, ddG, ddC or ddT). In Part A of the assignment you will identify the expected products in each of the four reactions and their expected lengths. In Part B, you will complete a table summarizing the reaction products and in Part C you will use the table constructed in Part B to read a portion of the insulin gene sequence.

Instructions

• Print the assignment.
• Complete Parts A, B and C.
• Return completed assignment by fax, e-mail, snail mail, or deliver to Science II.
• Assignment is worth 50 points total.

Part A. Tables of Reaction Products

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Instructions (2 pts. per line x 18 lines = 36 pts. total)

For each sequencing reaction fill in the expected reaction products (1 pt.) and the length of each product (1 pt.). Lengths are measured in bases and include the five base primer.

Use the examples shown in the dideoxy C and dideoxy G reactions as a guide.

Dideoxy C (ddC) Reaction

Example

Products	Length
5'AATTCATGTTC_______________ 3' 3'TTAAGTACAAGCAGTTAGTCGTGAAA 5'	11
5'AATTCATGTTCGTC____________ 3' 3'TTAAGTACAAGCAGTTAGTCGTGAAA 5'	14
Key: Primer = AATTC New strands = ATGTT and ATGTTCGT Dideoxynucleotide = C

Complete Table Of Other Reaction Products

Products	Length
3'AATTC_____________________ 5' 5'TTAAGTACAAGCAGTTAGTCGTGAAA 3'
3'AATTC_____________________ 5' 5'TTAAGTACAAGCAGTTAGTCGTGAAA 3'
3'AATTC_____________________ 5' 5'TTAAGTACAAGCAGTTAGTCGTGAAA 3'

Dideoxy G (ddG) Reaction

Example

Products	Length
3'AATTCATG__________________ 5' 5'TTAAGTACAAGCAGTTAGTCGTGAAA 3'	8
Key: Primer = AATTC New strand = AT Dideoxynucleotide = G

Complete Table Of Other Reaction Products

Products	Length
3'AATTC_____________________ 5' 5'TTAAGTACAAGCAGTTAGTCGTGAAA 3'
3'AATTC_____________________ 5' 5'TTAAGTACAAGCAGTTAGTCGTGAAA 3'

Dideoxy A (ddA) Reaction

Complete Table Of Reaction Products

Products	Length
3'AATTC_____________________ 5' 5'TTAAGTACAAGCAGTTAGTCGTGAAA 3'
3'AATTC_____________________ 5' 5'TTAAGTACAAGCAGTTAGTCGTGAAA 3'
3'AATTC_____________________ 5' 5'TTAAGTACAAGCAGTTAGTCGTGAAA 3'
3'AATTC_____________________ 5' 5'TTAAGTACAAGCAGTTAGTCGTGAAA 3'
3'AATTC_____________________ 5' 5'TTAAGTACAAGCAGTTAGTCGTGAAA 3'

Dideoxy T (ddT) Reaction

Complete Table Of Other Reaction Products

Products	Length
3'AATTC_____________________ 5' 5'TTAAGTACAAGCAGTTAGTCGTGAAA 3'
3'AATTC_____________________ 5' 5'TTAAGTACAAGCAGTTAGTCGTGAAA 3'
3'AATTC_____________________ 5' 5'TTAAGTACAAGCAGTTAGTCGTGAAA 3'
3'AATTC_____________________ 5' 5'TTAAGTACAAGCAGTTAGTCGTGAAA 3'
3'AATTC_____________________ 5' 5'TTAAGTACAAGCAGTTAGTCGTGAAA 3'
3'AATTC_____________________ 5' 5'TTAAGTACAAGCAGTTAGTCGTGAAA 3'
3'AATTC_____________________ 5' 5'TTAAGTACAAGCAGTTAGTCGTGAAA 3'
3'AATTC_____________________ 5' 5'TTAAGTACAAGCAGTTAGTCGTGAAA 3'

Part B. Summary of Reactions Products

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Instructions (11 pts. total):

For each reaction (ddA, ddC, ddG or ddT) place bars in the boxes corresponding to the lengths of the reaction products.

Length	ddA	ddC	ddG	ddT
26
25
24
23
22
21
20
19
18
17
16
15
14
13
12
11
10
9
8
7
6

Part C. Read The Sequence

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Instructions (4 pts. total):

Use the summary of reaction products table to read the sequence of the DNA strand.

Hint: Start at the bottom of the table

DNA SEQUENCE